Biocompatible substrate for facilitating interconnections between stem cells and target tissues and methods for implanting same

ABSTRACT

Disclosed herein are substrates for cell delivery to target tissues requiring treatment for various diseases that induce cell death, damage or loss of function. The substrates are configured to provide seeded cells, including stem cells, with a structural support that allows interconnection with and transmission of biological signals between the cells and the target tissue.

RELATED CASES

This application claims the benefit of U.S. Provisional Application Ser.Nos. 61/363,630, filed on Jul. 12, 2010 and 61/481,004, filed on Apr.29, 2011, the contents of each of which is expressly incorporated in itsentirety by reference herein.

BACKGROUND

1. Field of the Invention

The present application relates generally to substrates that facilitatethe administration of stem cells to target tissues in the context ofstem cell therapy as well as to tools for manipulating and implantingsuch substrates into a target tissue.

2. Description of the Related Art

The scope of human disease that involves loss of or damage to cells isvast and includes, but is not limited to, ocular disease,neurodegenerative disease, endocrine diseases, cancers, andcardiovascular disease. Cellular therapy involves the use of cells, andin some cases stem cells to treat diseased or damaged tissues. It israpidly coming to the forefront of technologies that are poised to treatmany diseases, in particular those that affect individuals who arenon-responsive to traditional pharmacologic therapies.

In fact, many diseases that are candidates for application of cellulartherapy are not fatal, but involve loss of normal physiologicalfunction. For example, ocular diseases often involve functionaldegeneration of various ocular tissues which affects the vision, andthus the quality of life of numerous individuals.

The mammalian eye is a specialized sensory organ capable of convertingincoming photons focused by anterior optics (cornea and lens) into aneurochemical signal. This process of phototransduction allows for sightby sending action potentials to higher cortical centers via the opticnerve. The retina of the eye comprises photoreceptors that are sensitiveto various levels of light and interneurons that relay signals from thephotoreceptors to the retinal ganglion cells. These photoreceptors arethe most metabolically active cells in the eye (if not the body), andare supported metabolically and functionally by retinal pigmentedepithelial cells (RPE). These RPE are positioned in a monolayer in theeye and are critical to vision.

Numerous pathologies can compromise or entirely eliminate anindividual's ability to perceive visual images, including trauma to theeye, infection, degeneration, vascular irregularities, and inflammatoryproblems. The central portion of the retina is known as the macula,which is responsible for central vision, fine visualization and colordifferentiation. The function of the macula may be adversely affected byage related macular degeneration (wet or dry), diabetic macular edema,idiopathic choroidal neovascularization, high myopia maculardegeneration, or advanced retinitis pigmentosa, among other pathologies.

Age related macular degeneration typically causes a loss of vision inthe center of the visual field. Macular degeneration occurs in “wet” and“dry” forms. Taken together, these diseases affect approximately 1.75million people in the U.S alone. The prevalence of those blinded by AMDis expected to increase to over 2.95 million by 2020. (See e.g.,Friedman, D S et al. Prevalence of age-related macular degeneration inthe United States. Arch Ophthalmol 2004; 122:564-72.) In the dry form,cellular debris (drusen) accumulates between the retina and the choroid,the blood supply of the outer retina, due to the inability of diseasedRPE cells of phagocytosing photoreceptor (PR) shed outer disc segments.Resulting hardened lipids (lipofuscin) impede the reciprocal exchange ofnutrients and waste products between the retina and choroid, and lead toPR death. In the more severe wet form, newly formed blood vessels fromthe choroid infiltrate the space behind the macula. The walls of thesenewly formed vessels are mechanically weak, and extremely susceptible torupture. Hemorrhage usually results in loss of vision extremely quicklycompared with dry AMD. In conjunction with the loss of functional cellsin the eye, the newly formed blood vessels are fragile and often leakblood and interstitial fluid, which can further damage the macula.

While diseases that cause damage to specific cells or tissues are clearcandidates for cellular therapy, there remains a need in the art formethods, substrates, and tools to improve the efficacy of cellulartherapy.

SUMMARY

Many tissues are structurally or functionally dynamic in that thetissues flex during normal function, are subject to fluid flow or othershear stresses, or have numerous specialized cell types in closejuxtaposition, thereby limiting the selection of target sites for celldelivery. As such, cellular therapy can require specific devices andmethods to administer cells to a target tissue that enhance the activityand beneficial effects of the administered cells at the target tissuefor an extended period of time. Depending on disease type, site ofadministration, advancement of pathology, and the type and time courseof integration required between graft and host, devices and methods forcellular therapy require specifications which optimize safety andefficacy of the specific therapeutic.

In several embodiments, there is provided a substrate for cellulartherapy to treat diseased or damaged ocular tissue, comprising anon-porous polymer having an apical and basal surface, wherein thesubstrate is configured to support a population of cells suitable forthe treatment of diseased or damaged ocular tissue, and wherein, uponimplantation into a subject, the substrate supports the population ofcells for a period of time sufficient to treat the diseased or damagedocular tissue.

In several embodiments, the substrate apical surface is substantiallyhomogeneous and suitable for the growth of cells thereon. In someembodiments, the thickness of the substantially homogeneous apicalsurface ranges from about 0.1 to about 4 microns. In one embodiment, thethickness of the substantially homogeneous apical surface is betweenabout 0.1 and about 0.5 microns. In some embodiments, the substantiallyhomogeneous apical surface is roughened or otherwise treated to allow apopulation of cells to have a non-planar surface to grow on. Thesubstantially homogeneous apical surface need not therefore becompletely uniform in all embodiments. Rather, several embodimentscomprise apical surfaces that are non-uniform, yet still substantiallyhomogeneous. In several embodiments, the thickness of the substantiallyhomogeneous apical surface for the growth of cells prohibits passage ofproteins larger than about 60 kDa through the substrate. The thicknesscan be altered in different embodiments depending on the cell type andthe size of proteins that are to be restricted (or allowed). Thetailored dimension allows for the passage of nutrients through thesubstrate (or the removal of metabolic byproducts through thesubstrate).

In several embodiments, the basal surface is inhomogeneous. In oneembodiment, the basal surface comprises a plurality of supportingfeatures juxtaposed with the apical surface. In one embodiment, a singlecontinuous support feature is present. In other embodiments, multiplesupport features are provided. The support features provide sufficientstructure to the substrate to allow for surgical implantation, but notso much rigidity that the substrate is kinked or otherwise creasedduring the implantation process. In several embodiments, the height ofthe supporting features ranges from about 3 μm to about 150 μm. In oneembodiment, the supporting features ranges from about 3 μm to about 8μm. In some embodiments, the substrate is oblong (e.g., longer thanwide) and in some such embodiments, the supporting features runlongitudinally along the long axis of the substrate. In this manner theyprovide structural rigidity along the long axis, but permit flexibilityalong the shorter axis. In other embodiments, the supporting featurescomprise columns of any appropriate geometric shape. In someembodiments, the substrate is round, in some embodiments, the substrateis square, and in some embodiments, the substrate is rectangular.

In one embodiment, the length and width of the substrate each range fromabout 0.3 mm to about 7 mm.

In several embodiments the outer edges and corners of the substrate arerounded, in order to minimize risk of damage to the substrate duringimplantation (e.g., by catching or snagging on tissue duringimplantation).

In some embodiments, the substantially homogeneous apical surfacefurther comprises a raised lip surrounding the surface, wherein theraised lip has a height ranging from about 10 to about 1000 microns anda width ranging from about 10 to about 1000 microns. In severalembodiments the lip serves not only to protect the cells growing on thesubstrate, but also to reduce trauma to the tissue during implantation.

In several embodiments, the non-porous polymer that comprises thesubstrate is non-biodegradable. In several embodiments, the non-porouspolymer is selected from the group of consisting parylene A, paryleneAM, parylene C, ammonia treated parylene, parylene X, parylene N, any ofthe foregoing polymers coated with polydopamine, any of the foregoingpolymers coated with matrigel, any of the foregoing polymers coated withvitronectin, and any of the foregoing polymers coated with retronectin.

In one embodiment, the non-porous polymer is oxygen-treated. In oneembodiment, the non-porous polymer is parylene C coated with one or moreof matrigel, vitronectin, and retronectin. In one embodiment,oxygen-treated polymers are coated with one or more of matrigel,vitronectin, and retronectin (or combinations thereof). In otherembodiments, alternatives to matrigel, vitronectin, and retronectin mayalso be used in place of or in addition to matrigel, vitronectin, andretronectin. Likewise, other modifying treatments (in addition to or inplace of oxygen-treatment) may be employed.

In other embodiments, the non-porous polymer is a biodegradable polymer.In some such embodiments, the biodegradable polymer is selected from thegroup consisting of PLGA, polyethylene glycol modified andpolycaprolactone.

In still additional embodiments, combinations of biodegradable andnon-biodegradable polymers are used.

In several embodiments, the substrates disclosed herein are configuredto support a population of retinal pigmented epithelial (RPE) cells. Inone embodiment, retinal pigmented epithelial cells are human embryonicstem cell-derived RPE cells. In several embodiments, after seeding asubstrate with RPE (or other stem cells), the cell-seeded substrate issuitable for implantation into the subretinal space of the eye of asubject, thereby treating an outer retinal degenerative disease. Inseveral embodiments, the substrates are suitable for implantation inorder to treat one or more outer retinal degenerative diseases(including, but not limited to) dry AMD, wet AMD, Stargardt's disease,and Leber's Congenital Ameurosis.

In several embodiments, there is also provided a substrate for cellulartherapy to treat diseased or damaged ocular tissue, comprising anon-porous polymer, wherein the substrate comprises a substantiallyhomogeneous apical surface for the growth of a population humanembryonic stem cell-derived RPE cells, wherein the thickness of thesubstantially homogeneous apical surface ranges from about 0.1 to about4 microns, wherein the substantially homogeneous apical surface isselected from the group consisting of parylene A, parylene AM, ammoniatreated parylene, and parylene C, wherein the substrate comprises aninhomogeneous basal surface comprising supporting features juxtaposedwith the substantially homogeneous apical surface, wherein theinhomogeneous basal surface comprises a polymer selected from the groupconsisting of parylene A, parylene AM, ammonia treated parylene, andparylene C, wherein one or more of the substantially homogeneous apicalsurface and the inhomogeneous basal surface is treated with one or moreof matrigel, poly-L-dopamine, vitronectin, or retronectin, wherein theheight of the supporting features ranges from about 3 μm to about 150μm; and wherein, upon implantation into a subject, the substratesupports the population of cells for a period of time sufficient totreat the diseased or damaged ocular tissue. In some embodiments, one ormore of the substantially homogeneous apical surface and theinhomogeneous basal surface are oxygen-treated.

In several embodiments, there is provided a substrate for cellulartherapy to treat diseased or damaged ocular tissue, comprising anon-porous biodegradable polymer, wherein the substrate comprises asubstantially homogeneous apical surface having a thickness from about0.1 to about 4 microns for the growth of cells, wherein the substratecomprises an substantially homogeneous basal surface juxtaposed with thesubstantially homogeneous apical surface, wherein the substrate isconfigured to support a population of cells suitable for the treatmentof diseased or damaged ocular tissue, and wherein, upon implantationinto a subject, the substrate supports the population of cells for aperiod of time sufficient to treat the diseased or damaged oculartissue.

In one embodiment, the substrate seeded with cells is suitable forimplantation into the subretinal space of the eye of a subject. In oneembodiment, the substrate is seeded with RPE cells, and whereinsubsequent to implantation, the RPE cells on the substrate functionallyinterdigitate with the outer segments of the photoreceptors of the eyeof the subject.

In several embodiments, there is provided a method of treating a subjecthaving outer retinal degenerative disease, comprising: surgicallypositioning a substrate comprising a non-porous polymer having an apicaland basal surface, wherein the apical surface is seeded with apopulation of cells into a position juxtaposed to the outer segments ofthe photoreceptors in the eye of the subject, thereby treating thedegenerative disease. In several embodiments, the substrate issurgically positioned in the sub-retinal space or adjacent to theepiretinal side of the retina of an eye of the subject.

In several embodiments, the cells seeded on the substrate are RPE cells.In several embodiments, the RPE cells functionally and/or metabolicallysupport damaged or diseased photoreceptors, thereby treating thedegenerative disease. In some embodiments, the RPE cells functionallyand metabolically support healthy photoreceptors as well. In severalembodiments, the RPE support the photoreceptors through metabolicallyfunctional interdigitation with the outer segments of thephotoreceptors.

In several embodiments, such methods are used to treat outer retinaldegenerative diseases which include, but are not limited to dry AMD, wetAMD, Stargardt's disease, and Leber's Congenital Ameurosis, andretinitis pigmentosa.

In several embodiments, there is provided a substrate for cellulartherapy to treat diseased or damaged ocular tissue comprising anon-porous polymer having a roughened apical surface topology for thegrowth of cells and an inhomogeneous basal surface comprising supportingfeatures juxtaposed with the roughened apical surface topology, whereinthe substrate is configured to support a population of cells suitablefor the treatment of diseased or damaged ocular tissue, and wherein,upon implantation into a subject, the substrate supports the populationof cells for a period of time sufficient to treat the diseased ordamaged ocular tissue. In several embodiments, the substrate isfabricated in a substrate frame comprising a plurality of substrates.

In some embodiments, the thickness of the substantially homogeneousapical surface ranges from about 0.1 to about 4 microns. In severalembodiments, the thickness of the substantially homogeneous apicalsurface for the growth of cells prohibits passage of proteins largerthan about 75 kDa through the substrate. In some embodiments, thesubstantially homogeneous apical surface further comprises a raised lipsurrounding the surface. In some such embodiments, the raised lip has aheight ranging from about 10 to about 1000 microns and a width rangingfrom about 10 to about 1000 microns. Other embodiments do not have alip. In some embodiments, the height of the supporting features rangesfrom about 3 μm to about 150 μm.

In several embodiments, the non-porous polymer is non-biodegradablewhile in other embodiments, the non-porous polymer is biodegradable. Insome embodiments, the biodegradable polymer comprises polyethyleneglycol modified polycaprolactone, PLGA, gelatin-modified silicone, or ananhydride polymer.

In several embodiments, the non-porous polymer is selected from thegroup consisting of parylene A, parylene AM, parylene C, ammonia and/oroxygen treated parylene C (for the purposes of adding functional groupsand roughening the surface), and parylene C treated with eitherpolydopamine, vitronectin, retronectin, or matrigel. In one embodiment,the non-porous polymer comprises parylene AM treated with polydopamine,and the inhomogeneous basal surface comprises parylene. In oneembodiment, the substrate is configured to support a population ofretinal pigmented epithelial (RPE) cells. In one embodiment, the retinalpigmented epithelial cells are human embryonic stem cell-derived RPEcells.

In several embodiments, the substrate is seeded on its substantiallyhomogeneous apical surface with cells selected from the group consistingof: RPE cells, RPE and photoreceptors, Müller glial cells, ganglioncells, a mixture of Müller glial cells and ganglion cells, cornealendothelial cells, a mixture of corneal endothelial cells and collagen,corneal epithelial cells, a mixture of corneal epithelial cells andcollagen, endothelial cells, pericytes, a mixture of endothelial cellsand pericytes.

In several embodiments, the cell-seeded substrate is implanted in thesubretinal space of the eye of a subject. In several such embodiments,the RPE cells interdigitate with the outer segments of thephotoreceptors of the eye of the subject. In some embodiments, the RPEcells interdigitate with the outer nuclear layer of the photorecptors.

In some embodiments, the cell-seeded substrate is implanted adjacent tothe epiretinal side of the retina of the eye of a subject.

In some embodiments, the cell-seeded substrate is implanted adjacent tocorneal tissue of the eye of a subject.

In several embodiments, the cell-seeded substrate is suitable forcellular therapy for treatment of dry AMD, for treatment of cornealdisease, for treatment of glaucoma, for treatment of diabeticretinopathy, for treatment of retinal vein occlusions, for treatment ofwet AMD, and/or for treatment of retinitis pigmentosa. Other oculardiseases may also be treated with such substrates.

In several embodiments, there is provided a substrate for preparingcells for cellular therapy to treat diseased or damaged ocular tissuecomprising a non-porous polymer comprising a substantially homogeneousapical surface for the growth of cells in an interconnected monolayer ofcells, wherein prior to delivery to a subject, the interconnectedmonolayer of cells is detached from the substrate and the monolayer isimplanted in the subject, thereby treating the diseased or damagedocular tissue.

In several embodiments, there is provided a substrate for cellulartherapy to treat diseased or damaged ocular tissue comprising anon-porous, permeable, non-biodegradable polymer selected from the groupconsisting of parylene A, parylene AM, ammonia etched parylene, andparylene coupled with polydopamine configured to form a roughened apicalsurface for the growth of cells, wherein the substantially homogeneousapical surface is coated with one or more of cyclic or lineararginine-glycine-aspartic acid or a synthetic growth matrix, and aninhomogeneous basal surface comprising supporting features juxtaposedwith the substantially homogeneous apical surface. In severalembodiments, the substrate is configured to support a population ofcells suitable for the treatment of diseased or damaged ocular tissueand, upon implantation into a subject, the substrate supports thepopulation of cells for a period of time sufficient to treat thediseased or damaged ocular tissue. In some embodiments, wherein thethickness of the substantially homogeneous apical surface ranges fromabout 0.1 to about 6 microns, and the height of the supporting featuresranges from about 3 μm to about 150 μm.

In several embodiments there is provided a system for preparing asubstrate for cellular therapy, comprising: a polymeric substrate framecomprising a plurality of individual substrates and a device fortemporarily maintaining the substrate frame in a fixed position within aculture vessel.

In several embodiments the individual substrates are capable of beingremoved individually from the substrate frame. In some embodiments, thedevice is configured to prevent growth of cells on at least one portionof each of the plurality of individual substrates, and some embodimentsthe device is configured to allow selective removal of an individualsubstrate from the substrate frame.

In some embodiments of the system, the polymeric substrate frame and theindividual substrates comprises a non-biodegradable polymer while inother embodiments, the polymeric substrate frame and the individualsubstrates comprises a biodegradable polymer.

In several embodiments the substrates are removed by cutting a portionof the substrate that connects the substrate to the substrate frame.Advantageously, several embodiments of the device configured to preventgrowth of cells comprises an aperture through which the portion of thesubstrate can be cut.

In several embodiments, there is provided a method of treating a subjecthaving outer retinal degenerative disease comprising surgicallypositioning an substrate as disclosed herein in a position juxtaposed tothe outer nuclear layer of the photoreceptors in the eye of the subject.In several such embodiments, RPE cells seeded on the substrate supportthe photoreceptors, thereby treating the degenerative disease.

In several embodiments, there is provided a three-dimensionalimplantable substrate cage for cellular therapy to treat outer retinaldegenerative disease. In some embodiments, the substrate cage comprisesan outer shell having one or more pores therein and configured to forman inner lumen which is configured to receive stem cells. In someembodiments, the pores are configured to retain the one or more types ofstem cells within the inner lumen while allowing an interaction betweenthe one or more types of stem cells and cells of the target tissue.

In several embodiments, there is provided a method for treating retinaldegeneration comprising culturing a plurality of stem cells, harvestingthe stem cells, deploying the cultured stem cells into athree-dimensional polymeric substrate cage, implanting the cage into atarget tissue.

In one embodiment, the outer shell of the substrate is polymeric. In oneembodiment, the substrate further comprises a reporting feature. In someembodiments the reporting feature comprises microelectromechanicalsystems (MEMS) technology. In some embodiments, the MEMS reportingfeature reports to a user regarding the viability of the cells housedwithin the substrate.

In one embodiment, the cells are RPE cells and the target tissue is themacula that has been damaged by age-related macular degeneration orother ocular disease. In one embodiment permeability of the substrate isdefined solely by thickness of the biocompatible substrate. In oneembodiment, the pores are between 0.5 and 1.5 μm. In one embodiment, theouter shell comprises polycaprolactone. In one embodiment, thepolycaprolactone shell further comprises one or more of PEG andArginine-Glycine-Asparagine. In one embodiment, the pores are generatedby exposing the polymer substrate to an aqueous media.

In several embodiments, there is provided a method of fabricating athree-dimensional substrate cage for cellular therapy comprisingpreparing a substrate for the growth of cells, generating pores withinthe substrate, and sterilizing the substrate. In one embodiment, one ormore types of stem cells are cultured on the substrate and athree-dimensional substrate cage is thereafter aligned and sealed,thereby containing the cells.

In several embodiments, there is provided a method of fabricating athree-dimensional substrate cage for cellular therapy comprisingpreparing two molds corresponding to top and bottom portions of thethree dimensional substrate cage and configured to form an inner lumenupon assembly, polymerizing a polymeric solution in each portion of themold, generating pores within the top and/or bottom portions of thesubstrate; and sealing the remaining portion or portions of thesubstrate cage with the substrate, thereby creating a three-dimensionalsubstrate cage for cellular therapy. In one embodiment, the substratecage is sterilized and sealed, and then cells are delivered into theinner lumen prior to implantation.

In one embodiment, the substrate cage is delivered to the subretinalspace of an individual having retinal degenerative disease, and thesubstrate cage retains the stem cells the substrate cage afterimplantation but also allows processes from the retained cells to passthrough the pores and interact with photoreceptors in the subretinalspace. In one embodiment, the substrate cage is delivered to thesubretinal space of an individual having retinal degenerative disease,and the substrate cage retains the stem cells after implantation butalso allows chemical and cellular interaction between the encapsulatedcells and the photoreceptors via the substrate cage pores. In oneembodiment, the interaction supports the photoreceptors, therebytreating the retinal degeneration. In one embodiment, theinterconnection further comprises one or more cells from the subretinalspace of the individual passing through the pores and interacting withthe substrate cage or the cells. In one embodiment, the substrate cageis implanted via an ab-interno surgical approach. In one embodiment, thesubstrate cage is implanted via an ab-externo surgical approach. In oneembodiment, the individual afflicted with retinal degeneration is amammal and in one embodiment, the mammal is a human. In one embodiment,the stem cells are deployed into the substrate cage immediately prior toimplantation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict general substrate structures in accordance withseveral embodiments disclosed herein.

FIGS. 2A-2B depict internal views of several embodiments disclosedherein. FIG. 2A depicts a side view of a substrate structure inaccordance with several embodiments disclosed herein. FIG. 2B depicts aninternal view of a substrate cage structure in accordance with severalembodiments disclosed herein.

FIG. 3 depicts a top cut-away view of a substrate in accordance withseveral embodiments disclosed herein.

FIG. 4 depicts a representation of an substrate cage with customizablechambers in accordance with several embodiments discloses herein

FIG. 5 depicts a representation of a patient's visual field used infabricating a customized substrate cage according to several embodimentsdisclosed herein.

FIG. 6 depicts an example of a scotoma map used to construct a customocular substrate.

FIG. 7 depicts a mold shell used in several embodiments of fabricationof a custom substrate.

FIG. 8 depicts an example of a custom fabricated substrate constructedaccording to methods disclosed herein.

FIG. 9 depicts the loading of cells according to several embodimentsdisclosed herein.

FIG. 10 depicts a cross-section of a substrate in accordance withseveral embodiments disclosed herein.

FIGS. 11A-11B depict the similarity between cultured hESC-derived RPE(FIG. 11A) cells and adult RPE cells (FIG. 11B).

FIGS. 12A-12B depict characteristics of cultured hESC-RPE. FIG. 12Ashows scanning electron microscopy of polarized hESC-RPE showing apicalspecialization and microvilli. FIG. 12B depicts phagocytic activity ofhESC similar to that of human native fetal RPE.

FIGS. 13A-13C depict visualization of post-implantation RPE cells. FIG.13A depicts rat fundus photograph showing PLGA-RPE one week afterimplantation in the subretinal space; FIG. 13B depicts an OCT scanrevealing the PLGA-RPE sheet (white arrow); FIG. 13C depicts the OCTimage through the non-transplant area.

FIGS. 14A-B depict hESC-RPE transplanted into the subretinal space ofRCS rats. FIG. 14A depicts nuclear staining (DAPI) of preservedphotoreceptors (ONL) in the transplanted area compared to the adjacentnon-transplanted area (FIG. 14B).

FIGS. 15A-15B depict growth of cells on various substrates in accordancewith several embodiments herein. FIG. 15A depicts hESC-RPE on PLGA byscanning electron microscopy (cross section). FIG. 15B shows growth ofhESC-RPE surface modified parylene.

FIGS. 16A-16B depict high resolution imaging SDOCT of the retina used inseveral embodiments disclosed herein.

FIGS. 17A-17B depict top and side views, respectively, of amulti-chambered substrate cage comprising a substrate used to delivercells in accordance with several embodiments herein. The substrate canbe either non-degradable or degradable, facilitating interaction betweencells in top and bottom chambers via defined material specifications. Insome embodiments degradation rate and thickness of the material may bedependent on the time required for cells in both chambers to beimplanted in the target location and make meaningful synapticconnections with, or allow for proper reciprocal nutrient exchangebetween, proximal cells.

FIGS. 18A-18D depict various embodiments of substrate layouts insubstrate frames.

FIGS. 19A-19B depict top views of various substrate shapes in accordancewith several embodiments disclosed herein.

FIG. 20A-20F depicts various substrates disclosed herein and relatedpermeability data. A side view of an asymmetric substrate having ahomogeneous apical surface 100 and an inhomogeneous basal surfacecomprising supporting structures in accordance with several embodimentsdisclosed herein is shown in FIG. 20A. Some embodiments further comprisea lip surrounding the apical surface 120 (see FIG. 20B). Someembodiments further comprise a coating on the cell growth surface (FIGS.20C-20D). Manipulation of the thickness of the substrate allows tuningof molecular size exclusion and diffusion (FIGS. 20E and 20F,respectively).

FIGS. 21A-21C depict various substrate embodiments disclosed herein.FIG. 21A depicts a scanning electron microscopic image of the planarapical surface of one embodiment of a substrate described herein. FIG.21B depicts a scanning electron microscopic image of the bottomsupporting surface of one embodiment of a substrate. FIG. 21C depicts agrowth of stem cells on the planar apical surface of a substrate.

FIG. 22A depicts an additional embodiment of substrate layout in asubstrate frame.

FIG. 22B depicts one embodiment of a device used to hold a substrateframe and cut individual substrates from the frame.

FIGS. 23A-23D depict histological results from implantation of a stemcell-seeded substrate implanted in a rat eye.

FIG. 24 depicts transmission electron microscopy of RPE cells implantedin the eye of a dystrophic rat at 9 days post-implantation.

FIG. 25 depicts transmission electron microscopy of RPE cells implantedin the eye of a dystrophic rat at 58 days post-implantation.Interdigitation is visible between the RPE microvilli and thephotoreceptor outer segment disks.

FIG. 26 depicts transmission electron microscopy of RPE cells implantedin the eye of a dystrophic rat at 58 days post-implantation.Interdigitation is visible between the RPE microvilli and thephotoreceptor outer segment disks.

FIG. 27 depicts scanning electron microscopy of RPE cells growing on theapical surface of a substrate in accordance with several embodimentsdisclosed herein.

FIGS. 28A-28C depict fluorescent immunohistochemistry images of RPEcells seeded on a polymeric substrate in accordance with severalembodiments disclosed herein.

FIGS. 29A-29C depict additional fluorescent immunohistochemistry imagesof RPE cells seeded on a polymeric substrate in accordance with severalembodiments disclosed herein.

FIG. 30 depicts optokinetic nystagmus (OKN) data collected from normal,untreated transgenic blind rats, and from treated transgenic blind ratsafter a bolus injection of RPE cells into the sub-retinal space of theeye of the rats.

FIG. 31 depicts OKN data collected from normal, untreated transgenicblind rats, and from treated transgenic blind rats after implantation ofa substrate seeded with RPE cells into the sub-retinal space of the eyeof the rats, as disclosed herein.

FIG. 32 depicts evidence of functional interdigitation (by detection ofrhodopsin) between photoreceptors and RPE cells seeded on a substrateand implanted into the eye of a transgenic blind rat.

DETAILED DESCRIPTION

Cellular therapy, the introduction of new cells into a tissue in orderto treat a disease, represents a possible method for repairing orreplacing diseased tissue with healthy tissue. Many approaches involveadministration of cells (e.g., stem cells) to a target tissue, whichoften yields low retention rates and decreased incidence of long-termpersistence of the transplanted (or implanted) cells. This may be due toa variety of factors, including cell washout and/or low cell survivalrates in the delivery media. However, some diseases do not require thedelivery or engraftment of the cells per se, but rather can requiregrowth factors, chemical signals, or other interactions with thedelivered cells. Several embodiments disclosed herein are directed totreating such diseases, and as such, comprise substrates configured todeliver the beneficial effects of cells (including physical, chemical,or other interaction) while retaining the cells within the substrate.

In particular, several embodiments relate to substrates into which stemcells are deposited in or on the substrate prior to administration ofthe substrate to a cell-therapy subject, and which function,post-administration, to retain the cells within or on the substratewhile simultaneously facilitating a physical interaction between thestem cells within or on the substrate and portions of the target tissue.In several embodiments in particular, stem cells deposited within or onthe substrate provide supportive effects for damaged or diseased cellsof a target tissue, including, but not limited to, physical cell-cellinteraction, release of nutrients or growth factors to the targettissue, attraction of other cell types, and the like. In severalembodiments, the beneficial effects include one or more of secretion ofgrowth factors which maintain the structural integrity of thechoriocappilaris endothelium and photoreceptors (e.g. PEDF and VEGF),suppression of immunosuppressive factors which aids in the immuneprivileged status of the eye (which helps to suppress immune cellinfiltration into the eye), secretion of neurotrophic factors, metabolic(e.g. exchange of glucose and fatty acids), functional benefits (e.g.delivery of retinol, phagocytosis of shed outer disc segments, and/orthe reisomerization and restoration of visual pigments afterphotobleaching), or support of neural activity. In some embodiments, thesupport of neural activity occurs via interdigitation of the cellswithin or on the substrate with target tissue cells. For example, in oneembodiment, apical microvilli of retinal pigmented epithelial cellswithin an implanted substrate interdigitate with host photoreceptors,thereby incurring a beneficial effect on the photoreceptors. In severalembodiments, the benefit is via synapse formation (e.g. PR/bipolarcell), or other physical or chemical support of general cell viability).In several embodiments, one or more of these benefits occurs whilepartially or fully retaining cell somas within or on the substrate. Insome embodiments, the cells from the host tissues project or infiltratethe substrate through the biological vias present in some embodiments ofthe substrate. For example, in some embodiments, new blood vessels orfibrous tissue protrude into the substrate. In some embodiments, theseprotrusions assist in anchoring the substrate, while in otherembodiments, other beneficial effects (e.g., nutrient delivery, bloodsupply) are realized. Thus, as used herein, the term “interaction” shallbe given its ordinary meaning and shall also refer to a one-way(implanted cells to target tissue or target-tissue to implanted cells)interconnect between cells or a two-way interconnect (both implantedcells to target tissue or target-tissue to implanted cells occur).

Further, in some embodiments, methods are provided for fabricating acustom substrate to treat a damaged or diseased tissue of an individualusing tailored stem cell therapy.

In several embodiments, porous substrates for improved cellular therapyare provided. In several embodiments, the substrates provided arenon-porous (e.g., do not have an orifice or via) but are permeable.Several such embodiments control the permeability of the substrate basedon changes in thickness of the substrate. In some embodiments,substrates are both permeable and porous, with the permeability andporosity facilitating the interconnections between the implanted cellsand the native cells. In several embodiments, the substrate isconfigured to receive cells and position the received cells in anoptimal manner to facilitate regeneration of a damaged or diseasedtarget tissue. In some embodiments, the substrates have specificcharacteristics (e.g., sizes, shapes, porosity) that retain the cellswithin the substrate, but simultaneously promote physical, chemical, orother interaction between the cells and target tissue. As used herein,the term “promote” shall be given its ordinary meaning and shall also beread to mean allow, enhance, permit, facilitate, foster, encourage,induce, and synonyms thereof. In some embodiments, the substrate isbiodegradable, while in others, a non-biodegradable substrate isprovided. In some embodiments the substrate is partly biodegradable andpartly non-biodegradable. In several embodiments, fullynon-biodegradable substrates are particularly advantageous, as theirpositioning relative to the implanted cells prevents access of immunecells into the transplant site and reduces risk of infection. Moreover,in several embodiments, a non-biodegradable substrate allows foridentification and explant of transplanted cells should removal orreplacement of the cells (e.g., additional “doses” of cells) berequired. In several embodiments, the substrates herein are fabricatedas a microelectromechanical system (MEMS). In several embodiments, thecells delivered to and retained within the substrate are stem cells. Inseveral embodiments the substrate, and the cells retained therein areused to treat the damage associated with a disease, such as oculardegeneration, cardiac disease, vascular disease, and the like.

Substrates

As discussed above, the variety of diseases that lead to damage or lossof function of particular cell types is vast and represents an area ofmedicine in need of treatment approaches that go beyond typical surgicalor pharmacological approaches. To address this need cellular therapyinvolves the use of cells, and in some cases fetal, umbilical cord,placenta-derived, adult, induced, or human embryonic stem cells and/ortheir partially or fully differentiated cellular derivatives to treatdiseased or damaged tissues via replacement or regeneration. In severalembodiments, substrates that improve the efficacy of cellular therapyare provided. As used herein, the terms “substrate” shall be given itsordinary meaning and shall also be used interchangeably with the term“implant” and/or “device”, though it shall be appreciated that someembodiments described herein do not require implantation per se (e.g.,those functioning as a “patch on a target tissue surface”). Thecontextual basis will make it clear to one of ordinary skill whether aparticular embodiment is to be implanted within a target tissue. Inaddition, as used herein, the term “deliver” shall be given its ordinarymeaning and shall also refer to the physical, chemical, or other type ofinteraction that the cells housed within the substrate provide to thetarget tissue without the release of cells from the substrate and/orengraftment of cells into the target tissue.

Types of Substrate

Based on the variety of diseases in which cellular therapy can beemployed, a variety of different types of substrates may beadvantageous, depending on the disease. In general, while many of thesubstrates disclosed herein inherently have three dimensions (e.g., alength, a width, and a height), some substrates disclosed herein aredesigned with particular attention being directed to one or more ofthese dimensions. For example, as discussed more fully below, severalembodiments are referred to as 3-D substrate cages. In such embodiments,the substrate is sufficient to allow formation of at least one interiorlumen. For example, in some embodiments, a cage structure with apurposefully designed 3-dimensional shape functions to provide one ormore lumens or cavities within the substrate which functions to retaincells within the structure after delivery to a target site (whilecontinuing to allow interactions between the cells and the targettissue). However, it shall be appreciated that the methods offabrication, implantation, and uses disclosed herein shall be applicableto of any variety of substrates, devices, or implants described herein,unless otherwise expressly specified.

In several embodiments, the substrate is asymmetrical and inhomogeneouswith distinct structural features on the apical and basal surfaces ofthe substrate. As used herein, the term “asymmetrical” and“substantially inhomogeneous” shall be given their ordinary meaning andshall also refer to substantially non-planar or variable surfaces.Likewise, as used herein, the term “substantially homogeneous” shall begiven its ordinary meaning and shall also refer to surfaces which arelargely or completely planar, or those which have minimally variablesurfaces. The term substantially homogeneous shall not exclude certainaccessory characteristics that cause a surface to not be completelyplanar. For example, the apical surface of the substrate is, in someembodiments, surrounded by a rim on the perimeter which is intended toprotect cell monolayer integrity from shear force imparted duringtransplantation and to inhibit lateral cell proliferation outside of theboundaries defined by substrate (see, e.g., 120 in FIG. 20B). While notcompletely planar, such embodiments are intended to be viewed assubstantially homogeneous. In several embodiments, the basal surface hasperiodic polymeric supports or columns providing mechanical stability tothe substrate thereby (1) facilitating handling during cell culturing,and loading onto custom tool and subsequent surgical implantation, and(2) shielding a thinner membrane and overlying cellular sheet frommechanical disruption from force imparted due to positive or negativeair pressure.

As used herein, the terms “3-dimensional” and “3-D” shall be given theirordinary meanings and shall also refer to those devices resembling acage (e.g., having one or more interior lumens or cavities). In light ofsuch variability in the design of substrates disclosed herein, thedisclosure below, unless otherwise specified shall be appreciated to beapplicable to any such variety of substrate.

Dimensions

Based on the variety of diseases in which cellular therapy can beemployed, and limitations of delivery of cells alone, severalembodiments provide substrates for improved cellular therapy. In severalembodiments, the disease to be treated or the cells to be targeteddefine, at least in part, the dimensions of the substrate. For example,in several embodiments substrates are dimensioned for implantation inparticular target tissues, while in other embodiments, substrates aredimensioned for placement on or near a target tissue.

In several embodiments, substrates disclosed here are utilized intreatment of ocular disease. In such embodiments, certain substratedimensions are utilized depending on the ocular tissue to be targeted.For example, a substrate to be implanted in the vitreal chamber wouldlikely differ in dimension from a substrate to be implanted in thesuprachoroidal space. In general, dimensions of certain ocular cavities,spaces, and tissue can be obtained from general knowledge of ocularanatomy. In certain embodiments, specific measurements are obtained froman individual to determine the specific dimensions required to fabricatea customized substrate.

In several embodiments, anchor features are fabricated into thesubstrate to allow secure and precise positioning of the substrate at atarget site. In some instances, once the substrate is in place and thecells within or on the substrate have established an interconnect withthe target cells, micro-movements of the substrate could damage and/orsever the interconnect, thereby reducing the therapeutic efficacy of thecells. Therefore, in several embodiments, anchor structures are attachedand/or built in to the substrates disclosed herein. For example, in someembodiments, one or more holes are provided that allow the substrate tobe sutured to target tissue. In some embodiments, a bioadhesive and/oran adhesive protein is used. In some embodiments, microhairs or aroughened surface of the substrate provide friction-based anchoring ofthe substrate. In some embodiments, MEMS features such as clamps orlatches are used to grasp or otherwise connect the substrate to thetarget tissue. In some embodiments, the target tissue is dimensionedsufficiently to securely hold a substrate without the need forspecialized anchors.

With reference to FIG. 1, certain general dimensions are provided for3-dimensional (e.g., cage) substrates according to several embodimentsdescribed herein. In several embodiments, the substrate 10 comprises asubstrate body 20 and a substrate tail 30. In some embodiments, thelength of the substrate tail D1 ranges from about 0.5 mm to about 5 mm.In some embodiments, the tail measures from about 0.5 to 1 mm, fromabout 1.0 to 1.5 mm, from about 1.5 to 2.0 mm from about 2.0 to 2.5 mm,from about 2.5 to 3.0 mm, from about 3.0 to 3.5 mm, from about 3.5 to4.0 mm, from about 4.0 to 4.5 mm, from about 4.5 to 5.0 mm, andoverlapping ranges thereof. In some embodiments, the substrate tailmeasures between about 0.7 to 1.3 mm, including 0.8, 0.9, 1.0, 1.1, and1.2 mm. In several embodiments, the substrate tail, the substrate body(below) or the total dimensions of the substrate as a whole are suchthat forceps may be used to grasp and manipulate the substrate. At thesame time, these dimensions are balanced with maintaining sufficientstructure to the substrate that it is not easily bent, kinked, orotherwise damaged during handling or implantation.

In several embodiments, the substrate body is generally circular (seee.g., FIG. 1A and FIG. 19A). However, in some embodiments (see e.g.,FIG. 1B and FIG. 19B), other shapes, including but not limited torectangles, squares, ovals, and cylinders are used. With reference toFIG. 1A, or other embodiments having a generally circular body, theradius of the substrate body measures from about 0.5 to about 5 mm. Insome embodiments, the radius measures from about 0.5 to 1 mm, from about1.0 to 1.5 mm, from about 1.5 to 2.0 mm from about 2.0 to 2.5 mm, fromabout 2.5 to 3.0 mm, from about 3.0 to 3.5 mm, from about 3.5 to 4.0 mm,from about 4.0 to 4.5 mm, from about 4.5 to 5.0 mm, and overlappingranges thereof. In some embodiments, the substrate body radius measuresbetween about 0.7 to 1.3 mm, including 0.8, 0.9, 1.0, 1.1, and 1.2 mm.

Similar dimensions are used in several embodiments not having a roundedor circular body. With reference to FIG. 1B, D1A represents the lengthof the substrate, and in some embodiments, ranges from about 0.5 toabout 5 mm. In some embodiments, the length measures from about 0.5 to 1mm, from about 1.0 to 1.5 mm, from about 1.5 to 2.0 mm from about 2.0 to2.5 mm, from about 2.5 to 3.0 mm, from about 3.0 to 3.5 mm, from about3.5 to 4.0 mm, from about 4.0 to 4.5 mm, from about 4.5 to 5.0 mm, andoverlapping ranges thereof. Dimension D2A represents the width of thesubstrate and may have similar measurements as D1A above.

With reference now to FIG. 2A, which is a side view depicting the3-dimensional structure of several embodiments disclosed herein,dimensions D3 and D4 represent the thickness of the substrate cage wall30. D3 and D4 have the same dimension in some embodiments, while inother embodiments, the dimensions vary between the two. In someembodiments, D3 and/or D4 measure between about 1.0 and 5.0 microns. Insome embodiments, the thickness of the wall ranges from about 0.5 to 1μm, from about 1.0 to 1.5 μm, from about 1.5 to 2.0 μm, from about 2.0to 2.5 μm, from about 2.5 to 3.0 μm, from about 3.0 to 3.5 μm, fromabout 3.5 to 4.0 μm, from about 4.0 to 4.5 μm, from about 4.5 to 5.0 μm,and overlapping ranges thereof. In some embodiments, the thickness of D3and/or D4 is ranges from about 1.5 to 2.5 μm, including 1.6, 1.7. 1.8,1.9, 2.0, 2.1, 2.2, 2.3, and 2.4 μm. In one embodiment, D3 is maximallyabout 7 μm, which allows microvilli to penetrate the vias 60 fully. Inone embodiment, D4 is greater than D3 in order to provide structuralrigidity to the substrate cage. In some embodiments, one or moresurfaces of the substrate cage is fabricated in a flexible “accordion”shape, to allow the substrate cage to expand upon delivery of cells tothe lumen of the substrate cage. In some embodiments this expansion ismade possible by using a highly elastomeric polymer. In someembodiments, the cell delivery solution comprises a larger overallvolume than the volume of cells that are desired to be retained withinthe substrate cage. As such, the accordion shape of some embodiments,allows the substrate cage to expand to accommodate this excess fluid andthen retract as the excess fluid passes through the pores of thesubstrate cage.

As shown in FIG. 2A, the lumen 50 is an open space within the outershell of the substrate cage that houses the cells that will provide atherapeutic effect to the target tissue. Depending on the cell type tobe delivered, the quantity of cells to be housed within the substratecage, and other limiting physical parameters of the target tissue, theheight of the lumen D6 can range from about 4 μm to about 75 μm. In someembodiments, D6 measures from about 4 to 10 μm, from about 5 to 20 μm,from about 10 to 30 μm, from about 25 to 40 μm, from about 30 to 50 μm,from about 45 to 60 μm, from about 50 to 75 μm, from about 65 to 75 μm,or overlapping ranges thereof. In several embodiments, D6 ranges fromabout 10 to about 50 μm. In some embodiments, the height D5 of the lumen50 is the same as the height of the membrane 70 that allows celldelivery to the lumen, but prevents cellular backflow. However, in someembodiments, depending on the overall shape of the substrate may vary,such that a first portion does not have the same dimension as a secondportion.

The total height of the substrate cage D5 is a function of D3, D4 andD5, as well as any characteristics of the target tissue that need to beaccounted for in the fabrication of the substrate cage. In someembodiments, D5 ranges from about 6 to about 85 μm, including 5 to 20μm, 20 to 30 μm, 30 to 40 μm, 40 to 50 μm, 50 to 60 μm, 60 to 70 μm, 70to 85 μm and overlapping ranges thereof. In some embodiments, thedimensions are adjusted to account for the location of the targettissue. For example, if the target tissue is frequently under load(e.g., pressure, contractile, etc.) but presents a small area suitablefor implantation, the substrate cage can be fabricated with thickerwalls and a suitable overall height (larger D3 and/or D4 relative toD5). As a result, D6 would be smaller. In other embodiments, walls ofthe substrate cage can be fabricated to maximize D6 relative to D5.Thus, in several embodiments, the dimensions of the substrate cage aretailored to the target tissue generally, or in some embodiments, to thetarget tissue dimensions of a particular individual.

With reference to FIGS. 18A and 22A, in several embodiments, a pluralityof substrates are positioned within a culture vessel in order to allowfor the concurrent seeding of cells across the plurality of substrates.In several embodiments, the diameter D8 is designed to be slightlysmaller than commercially available cell culture dishes adequate forcGMP scale up such that the substrate frame fits snuggly in the dish(e.g., fit to reduce/minimize movement of the substrate). Based on thediameter D8 (or width, if not circular) of the substrate frame culturevessel, the number of substrates that are concurrently seeded with cellswill vary. In some embodiments, and depending on culture dish diameter,D8 is about 14-15 cm, about 9-10 cm, or about 4-5 cm. In someembodiments, D8 is less than about 4 cm, including 3, 2, 1, and 0.5 cm.In several embodiments, multiwell culture vessels are used. For example,in several embodiments 6-well, 12-well, 24-well, or 48-well plates areused. In some embodiments, 96-well plates are used. In severalembodiments, the depth of the wells ranges from about 1 to about 1.7 mm,including about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 mm. In several embodiments,the diameter of the wells (which can be optimized based on the number ofsubstrates to be cultured in the well) ranges form about 3 to about 20mm, including about 3 to about 5 mm, about 5 to about 7 mm, about 7 toabout 9 mm, about 9 to about 11 mm, about 11 to about 13 mm, about 13 toabout 15 mm, about 15 to about 17 mm, about 17 to about 19 mm, about 19to about 20 mm, and overlapping ranges thereof. In several embodiments,the height of the sidewalls ranges from about 10-20 mm, including about10 to about 12 mm, about 12 to about 14 mm, about 14 to about 16 mm,about 16 to about 18 mm, about 18 to about 20 mm, and overlapping rangesthereof. Certain embodiments, of such culture vessels are commerciallyavailable with established well dimensions.

In some embodiments, a culture vessel that is rectangular or square isused in order to maximize the culture area of the vessel. In someembodiments, and especially with regard to rectangular or square dishes,a custom sized (e.g., not commercially available) culture vessel isdesigned and fabricated. Specifications of these custom dishes such aswidth and length are defined by multiple variables, including, but notlimited to, (1) the minimum number of substrates or custom tool headsrequired to provide adequate sampling at final release testing to showlot-to-lot consistency, (2) finalized substrate shape (either circular(FIG. 19A) or rectangular (FIG. 19B)) and associated lateral dimensions(D9-D11 with regard to circular embodiment; D10, D12, D13 with regard tooblong or rectangular embodiment with rounded edges, or a oval shapedhybrid of the two), etc.

Based on the dimensions of the culture vessel, and the target tissue forthe substrate, the dimensions of the substrates may vary. As discussedin more detail below and shown generally in FIG. 19A, in severalembodiments, there is provided a substantially inhomogeneous substratehaving a circular body and a tail. The dimensions of this type ofsubstrate, while overlapping with the 3-dimensional substrate cagesdescribed above, are specifically designed with the strength,durability, and manipulability of substrates that do not have anaturally increased stability by being formed in a cage-like structureor do not require a lumen. In some embodiments, a fully planar substrateis used. In some embodiments, both top and bottom surfaces arenon-planar.

As such, the diameter D9 of such inhomogeneous substrates ranges fromabout 1 mm to about 8 mm, including about 1 to about 2 mm, about 2 toabout 3 mm, about 3 to about 4 mm, about 4 to about 5 mm, about 5 toabout 6 mm, about 6 to about 7 mm, about 7 to about 8 mm, andoverlapping ranges thereof. In certain embodiments, diameters of about 3to about 5 mm are used, including 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4,4.6, 4.8, and 5.0 mm.

Tail (or handle) width D10 ranges, in several embodiments, from betweenabout 0.1 mm to about 6 mm, including about 0.2 to about 1.0 mm, about1.0 to about 2.0 mm, about 2.0 to about 3.0 mm, about 3.0 to about 4.0mm, about 4.0 to about 5.0 mm, about 5.0 to about 6.0 mm, andoverlapping ranges thereof.

Tail (or handle) length D11 ranges, in several embodiments, from betweenabout 1 mm to about 20 mm, including about 1 to about 2 mm, about 2 toabout 3 mm, about 3 to about 4 mm, about 4 to about 5 mm, about 5 toabout 6 mm, about 6 to about 7 mm, about 7 to about 8 mm, about 8 toabout 9 mm, about 9 to about 10 mm, about 10 to about 11 mm, about 11 toabout 12 mm, about 12 to about 13 mm, about 13 to about 14 mm, about 14to about 15 mm, about 15 to about 16 mm, about 16 to about 17 mm, about17 to about 18 mm, about 18 to about 19 mm, about 19 to about 20 mm, andoverlapping ranges thereof.

Similar tail width and length are used in oblong (e.g., oval or largelyrectangular) substrates, as depicted generally in FIG. 19B. Width D12 ofsuch substrates ranges, in several embodiments between about 0.2 andabout 7 mm, including from about 0.2 to about 0.4 mm, about 0.4 to about0.6 mm, about 0.6 mm to about 0.8 mm, about 0.8 mm to about 1.0 mm,about 1.0 to about 2.0 mm, about 2.0 to about 3.0 mm, about 3.0 to about4.0 mm, about 4.0 to about 5.0 mm, about 5.0 to about 6.0 mm, andoverlapping ranges thereof.

The length D13 of such oblong substrates ranges, in several embodiments,from between about 0.5 mm to about 9 mm, including about 0.5 to about1.0 mm, about 1.0 to about 2.0 mm, about 2.0 to about 3.0 mm, about 3.0to about 4.0 mm, about 4.0 to about 5.0 mm, about 5.0 to about 6.0 mm,about 6.0 to about 7.0 mm, about 7.0 to about 8.0 mm, about 8.0 to about9.0 mm and overlapping ranges thereof. In some embodiments, length D13ranges from about 4 to about 7 mm, including 4.2, 4.4, 4.6, 4.8, 5.0,5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.8, and 7.0 mm.

With reference to FIG. 20, certain general cross-sectional dimensionsare provided for inhomogeneous substrates with and without a perimeterlip (FIG. 20B). Embodiments employing either of these cross-sectionalstructures can be used with commercially available cell culture dishes,or other types of dishes such as those described above. In severalembodiments, the substrate has a homogeneous apical layer 100 whichprovides a surface for cellular growth. In several embodiments, thethickness D14 of the apical cell growth surface 100 is less than about 4μm thick. In some embodiments, the thickness ranges from about 0.05 toabout 0.1 μm, from about 0.1 to about 0.2 μm, from about 0.2 to about0.5 μm, from about 0.5 to about 3.5 μm, from about 0.5 to about 3.0 μm,from about 0.7 to about 3.0 μm, from about 1.0 to about 3.0 μm, fromabout 1.5 to about 2.5 μm, from about 1.8 to about 2.2 μm, andoverlapping ranges thereof. In some embodiments, the thickness rangesfrom between about 0.5 and 1.0 μm, including 0.6, 0.7, 0.8, and 0.9 μm.In certain embodiments, a thicker apical cell growth surface is used.For example, in some embodiments, the apical cell growth surface rangesfrom about 2 to about 3 μm, about 3 to about 4 μm, about 4 μm to about 5μm, about 5 μm to about 6 μm, and overlapping ranges thereof. Dependingon the target tissue, and on the specific region of the target tissue,and the cell type, thicker or thinner apical surfaces may be also used.As shown, the apical cell growth surface 100 is paired with thickersupports 110 on the basal surface. The supports 110 provide in someembodiments, mechanical rigidity, which aids in supporting the fullapical cell growth surface during handling and surgical insertion. Inthose embodiments wherein the cells growing on the surface are RPEcells, after surgical insertion, the apical cell growth surface (and theRPE cells) will be juxtaposed with the photoreceptors in the eye,thereby serving to support the health of the photoreceptors. In severalembodiments, the supports also inhibit lateral growth of the cells andpossible extension of growth onto the basal side of the substrate. Thesupports, in several embodiments also inhibit cell growth into thevitreous via mechanical disruption of retinal integrity. (The latterresults in a complication known as proliferative vitreo-retinopaty(PVR)). Therefore, such embodiments have a homogeneous apical surfaceconducive to cell growth paired with a basal surface having aninhomogeneous, but periodic, topology defined by the size and pitch ofthe supports. In some embodiments, the supports are columnar in shape,while in other embodiments, other shapes are used (e.g., cuboidal). Insome embodiments a rim is employed on the apical surface to inhibitlateral growth and protect from shear force during surgicalimplantation.

As shown in FIGS. 20A and 20B, the basal surface supports 110 range intotal height D15 (as measured from the apical cell growth surface layerto the termination of the support) between about 3 μm and about 15 μm,including about 3 to about 5 μm, about 5 to about 7 μm, about 7 to about9 μm, about 9 to about 11 μm, about 11 to about 13 μm, about 13 to about15 μm, and overlapping ranges thereof. In some embodiments, the supportsrange in height from between about 5 μm to about 8 μm, including 5.2,5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8, and 8.0μm. Depending on the embodiment, larger supports (e.g., greater height)or smaller (e.g., lesser height) may also be used. For example, inseveral embodiments, the height of the supporting features ranges fromabout 5 μm to about 500 μm, including about 5 μm to about 50 μm, about50 μm to about 100 μm, about 100 μm to about 150 μm, about 150 μm toabout 200 μm, about 200 μm to about 300 μm, about 300 μm to about 400μm, about 400 μm to about 500 μm, and overlapping ranges thereof.Likewise, in several embodiments the height of the supporting featuresranges from about 1 μm to about 5 μm, including about 1 μm to about 1.5μm, about 1.5 μm to about 2.0 μm, about 2.0 μm to about 2.5 μm, about2.5 μm to about 3.0 μm, about 3.0 μm to about 3.5 μm, about 3.5 μm toabout 4.0 μm, about 4.0 μm to about 5.0 μm, and overlapping rangesthereof.

In several embodiments, the apical and basal surfaces comprise differentmaterials. For example, in one embodiment, the apical cell growthsurface may be made of first material that is more conducive to cellgrowth, while the basal surface is made of a second (or modified firstmaterial) that imparts strength and/or durability to the substrate as awhole. In some embodiments, the apical surface comprises a coating orthin layer of deposited material 100′ that assists in cell growth and/oradherence. (see e.g., FIGS. 20C and 20D). In several embodiments, thislayer is between about 10 and 100 nm in thickness. In some embodiments,the layer is between about 20 to 30 nm, between about 30 to 40 nm,between about 40 to 50 nm, between about 50 to 60 nm, between about 60to 70 nm, between about 70 to 80 nm, between about 80 to 90 nm, betweenabout 90 to 100 nm, and overlapping ranges thereof.

In several embodiments, the layer 100′ comprises parylene AM. In severalembodiments the layer comprises ammonia treated parylene C. In severalembodiments, the layer comprises parylene C and polydopamine. In severalembodiments, parylene AM, parylene C, ammonia and/or oxygen treatedparylene C (for the purposes of adding functional groups and rougheningthe surface), and parylene C treated with either polydopamine,vitronectin, retronectin, or matrigel are used. In several suchembodiments, the second layer (100 in FIGS. 20A-20D) comprises a blendof cyclic and linear arginine-glycine-aspartic acid residues. In severalembodiments, the second layer comprises a synthetic cell growth matrix(e.g., SYNTHEMAX™ by Corning). In one embodiment, the inhomogeneousbasal surface comprises parylene C, with a parylene AM layer as thesubstantially homogeneous apical surface. In one embodiment, theparylene AM is coated with one or more of matrigel, vitronectin,retronectin, poly-L-dopamine. In one embodiment the substrate comprisesparylene C, which is coated and/or treated directly. In severalembodiments the poly-L-dopamine coating is generated by reactingPEG-(N-Boc-L-DOPA)₂) with cyclic Arginine-Glycine-aspartic in anoxidative aqueous media (such as sodium periodate (NaIO₄) to generate apoly-L-dopa coating. Alternatively, PEG-(N-Boc-L-DOPA)₂) is reacted withRGD-L-DOPA in an oxidative aqueous media (such as sodium periodate(NaIO₄) to generate a poly-L-dopa coating. In several embodiments thesubstrate comprises parylene C, which is coated and/or treated directly.In one embodiment, the substrate is coated with Matrigel, retronectin,vitronectin, equivalents thereof, and/or combinations thereof. Moreover,in several embodiments, a coating on the surface of the substrate isused in conjunction with another surface treatment method, such as, forexample oxygen treating the substrate surface (disclosed in more detailbelow).

Alternative embodiments, a non-limiting example of which is shown inFIG. 20B, further comprise a substrate lip 120 lining the perimeter ofthe apical cell growth surface 100 that functions to shield cellsgrowing on the apical surface from fluid shear stress during culturingof the cells, manipulation of the substrate, and/or during or aftersurgical implantation into a target tissue (e.g., the eye). The lipfurther functions to define a boundary to control growth of the cells onthe surface; thereby preventing the growth of the cells from the apicalsurface (cell growth surface 100) to the basal side of the substrate.Moreover, the substrate lip 120 limits disruption of monolayer integrityduring the separation of an individual substrate from an array ofsubstrates (e.g., those shown in FIG. 18A or 22A). In severalembodiments the lip has a width D18 ranging from between about 10 μm toabout 1500 μm (1.5 mm). In some embodiments, the width ranges from about10 to about 100 μm, from about 100 to about 200 μm, from about 200 toabout 300 μm, from about 300 to about 400 μm, from about 400 to about500 μm, from about 500 to about 600 μm, from about 600 to about 700 μm,from about 700 to about 800 μm, from about 800 to about 1000 μm, fromabout 1000 to about 1100 μm, from about 1100 to about 1200 μm, fromabout 1200 to about 1300 μm, from about 1300 to about 1400 μm, fromabout 1400 to about 1500 μm, and overlapping ranges thereof.

In several embodiments the lip has a height D19 ranging from betweenabout from between about 10 μm to about 1500 μm (1.5 mm). In someembodiments, the height ranges from about 10 to about 100 μm, from about100 to about 200 μm, from about 200 to about 300 μm, from about 300 toabout 400 μm, from about 400 to about 500 μm, from about 500 to about600 μm, from about 600 to about 700 μm, from about 700 to about 800 μm,from about 800 to about 1000 μm, from about 1000 to about 1100 μm, fromabout 1100 to about 1200 μm, from about 1200 to about 1300 μm, fromabout 1300 to about 1400 μm, from about 1400 to about 1500 μm, andoverlapping ranges thereof. In some embodiments, the height ranges frombetween about 20 to about 300 μm, including about 20 to about 50 μm,about 50 to about 100 μm, about 100 to about 150 μm, about 150 to about200 μm, about 200 to about 250 μm, about 250 to about 300 μm, andoverlapping ranges thereof.

As shown in FIGS. 21A-21C, such substrates having an apical cell growthsurface and basal structural support surface can be fabricated by themethods disclosed herein and support cellular growth. FIG. 21A depicts ascanning electron microscopy image of the apical cell growth surface ofan substrate in accordance with the above description. As shown, theapical cell growth surface is homogeneous, while, as shown in FIG. 21B,the basal surface is inhomogeneous, comprising multiple supportstructures. Further, as shown in FIG. 21C, the apical cell growthsurface is suitable for the growth of cells, e.g., stem cells (shown areH9 hESC-RPE cells proliferating on a parylene growth surface).

As discussed above, the particular tissue that is damaged or diseasedand is to be targeted with a cell-loaded substrate may define thedimensions or structural features of the substrate. For example, asubstrate used to target cells to the surface of an individual's livercould be fabricated with much larger dimensions than those describedabove. Similarly, a substrate fabricated to target the cardiac tissue ofa patient could also be designed with larger overall dimensions.However, some substrates, such as those to target neural tissues, maybenefit from dimensions more similar to those described above. Given theanatomical knowledge of those skilled in the art, dimensions for varioustarget tissues can be readily obtained, or as discussed herein,specifically measured for a certain individual.

Porosity and Permeability

With reference to FIGS. 1 and 2, in several embodiments, the substratecages comprise porous materials. In some embodiments, the material is apermeable material. As shown, pores 60 are present in the outer shell ofthe substrate cage. In some embodiments, pores are present on the top 30a and bottom 30 b surfaces of the substrate cage. As used herein, theterm pore shall be given its ordinary meaning and also refer to“biological vias”, in the sense that they function as passageways toallow the physical, chemical, or other types of interactions and/orinterconnections described herein. The terms “pore” and “via” shall beread as interchangeable unless contextually indicated otherwise. In someembodiments, the vias on the top side of the substrate cage allow thepassage of apical microvilli from cells contained within the substratecage, while vias on the bottom side provide support for the cell bodies.In some embodiments, the density of pores on the top and bottom sides issimilar, while in other embodiments, the pore density is different.

In several embodiments, pore density ranges between about 1×10⁶ and2.5×10⁹ pores per cm². In some embodiments, pores have a distancebetween them of approximately 0.2 microns and about 2 microncenter-to-center pitch. Greater or lesser spacing and pitch is used inother embodiments. In several embodiments, the parameters describedabove (pore diameters, density, and substrate thickness) affect thehydraulic conductivity and diffusion rate of nutrients andmacromolecules across the substrate. In one embodiment, net flux acrossboth substrates is zero. As such, in several embodiments, the bottomsurface has a higher pore density compared with the top. To this end adual layer photolithographic process flow allowing for decreasedallowable effective pore diameter defined by sacrificial layer thicknesscan be employed for fabrication of the basal substrate. In some suchembodiments, a first layer of material is fabricated with passages thatcommunicate with either surface of the layer. This results, incross-section, in a “block-like” pattern. See e.g., FIG. 10. There aftera second layer is fabricated, again with similar passages. In order tomaintain the desired pore diameter, the second layer isphotolithographically laid onto the first layer with a known gap size 60that corresponds to the desired size of the biological via.

In still other embodiments, vias are present only on one of the sides,e.g., top or bottom. In some embodiments, although not expressly shownin the Figures, the apical and/or basal surface further comprises aseparate substrate material that is annealed to the apical or basalsurface during the fabrication process. In several embodiments, the viasrange in diameter from about 0.5 to about 10 μm. Pore diameter may varydepending on the cell type to be housed within the substrate cage, thesite of implantation, or the target tissue. In several embodiments, thepore diameter ranges from about 1 to 3 μm, 3 to 5 μm, 5-7 μm, 7 to 10μm, or overlapping ranges thereof. In several embodiments, the porediameter ranges from about 0.5 to about 1.5 μm, including 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, and 1.4 μm. While in several embodiments, thepore diameter is sufficiently large to allow cellular process to reachout of the substrate cage to the target tissue, the pore diameter is notlarge enough to allow the cell itself to escape the substrate cage.Thus, via diameter serves to retain the cells within the substrate cage,but allows the therapeutic effect of the cells to reach the targettissue, whether this be a physical (e.g. between a cellular process andthe target tissue), chemical, or other type of interaction. However, insome embodiments, substrate cages are designed to let at least a portionof the cells within the substrate cage escape.

In several embodiments, the pore diameter is varied across the surfaceof the substrate cage during the fabrication process. In severalembodiments, pore size may be varied to allow cells at a certainposition in the lumen of the substrate cage to escape while other cellsare retained within the substrate cage. In some embodiments, thesubstrate cage is fabricated with multiple chambers, and pore size mayvary depending on the chamber the pore is in communication with. Forexample, a first chamber may house a first cell type to be retainedwithin the substrate cage and provide a therapeutic effect to the targettissue. In such embodiments, the first chamber would be fabricated witha pore size that retained the cells within the substrate cage. See, forexample FIG. 2B chamber 50 a and pore 60 a. However, a second cell typemay be housed within a second chamber, the second cell type providing anancillary effect to the first cell type and/or the target tissue uponescape from the substrate. Thus, in such embodiments, the second chamberwould be fabricated with pores of a diameter sufficient to allow thesecond cell type to escape the substrate cage. See, for example, FIG.2B, chamber 50 b and pore 60 b. In other embodiments, different chambersmay house drugs or other ancillary agents, and are therefore fabricatedwith a porosity defined by the required or desired release rate of thedrug or agent. In some embodiments, additional chambers are not requiredfor a drug or ancillary agent. The drug or ancillary agents are used tosupport the viability of the cells, promote the interaction of the cellswith the target tissue, inhibit or promote vascularization of thesubstrate or tissue near the substrate (depending on the target tissue)or other additional effects that potentiate or otherwise enhance thetherapeutic effects of the cells on the target tissue.

With reference to FIG. 4, different chambers 50, 50 a, 50 b, and 50 cmay also be fabricated in a concentric or semi-concentric manner. Theindividual chambers are created by dividers 90, 90 a, and 90 b. Inseveral embodiments, the dividers comprise the same material as the bodyof the substrate cage. In several embodiments, dividers are constructedof a different material from the body of the substrate cage. In someembodiments, the dividers comprise wires through which an electricalcurrent may be passed to create and seal off a chamber from theremainder of the lumen of the substrate cage. In other embodiments, thematerials that generate the chambers may be altered to allow forvisualization of the substrate cage in situ. For example, 90, 90 a, and90 b, may comprise a ring of chromium or other compound that allowsvisualization. These structures may also function as connectors forchemical sensors that allow reporting of information regarding theenvironment around the substrate (below). Further, they could functionas a mechanism to secure an apical and basal portion of an substrate toone another, either before or after the seeding of the cells.

In several embodiments, the substrates are non-porous, e.g., no orificeor via exists that passes through the thickness of the substratematerial. While lacking a specific passageway for passage of nutrientsor cell processes, non-porous substrates have a permeability that can bemanipulated based on the thickness of the material used. For example,the thickness of the substrate material allows the substrate to act as amolecular sieve, keeping certain proteins of a certain size from passingthrough the substrate, while allowing passage of proteins of other sizesthrough the substrate. See FIGS. 20E and 20F that depict data related tothe diffusion of molecules (dextran) through substrates ranging from0.15 to 0.80 μm in thickness. Diffusion coefficients (related tothickness) range from between about 10⁻¹⁰ and 10⁻¹³ cm²/second. In someembodiments, greater or lesser rates of diffusion can be achieved bymanipulating the thickness of the substrate.

As discussed above, several asymmetrical, inhomogeneous substrateembodiments (those with an apical cell growth surface and aninhomogeneous basal surface) are non-porous, but are permeable. Acertain degree of permeability of the substrate material is necessary tosupport the cells grown on the substrate such that they are bothmetabolically and functionally viable over time (e.g., both in cultureand post-implantation). For example, in several embodiments, parylenesubstrates having a thickness (e.g., D14 in FIGS. 20A-20) less thanabout 0.80 μm have a molecular weight exclusion limit of about 70-75kDa. Such a substrate would exclude proteins or molecules larger thanabout 70-75 kDa, which would allow for the passage of most proteinspresent in the bloodstream that would be necessary to support cells onthe substrate after implantation. In some embodiments, greater or lessermolecular exclusion can be achieved by manipulating the thickness of thesubstrate (e.g., exclusion limits ranging from about 25 kDa to about 150kDa, including about 25 to about 30 kDa, about 30 to about 35 kDa, about35 to about 40 kDa, about 40 to about 45 kDa, about 45 to about 50 kDa,about 50 to about 55 kDa, about 55 to about 60 kDa, about 60 to about 65kDa, about 65 to about 70 kDa, about 70 to about 75 kDa, about 75 toabout 100 kDa, about 100 to about 125 kDa, about 125 to about 150 kDa,and overlapping ranges thereof.

While it is appreciated in the art that delivery of nutrients toimplanted (and existing cells) is important for the viability of thecells, it is particularly advantageous that certain embodiments of thesubstrates disclosed herein do not require a pore or an orifice toprovide such nutrients. For example, wet AMD involves anomalousneovascularization by vessels that have mechanically weaker walls. Thisfragile vasculature risks rupture, subsequent hemorrhage, and the rapidloss of vision due to cell death. Such rupture could be compounded bythe implantation of substrates having pores, through which such fragilevessels could grow. While dry AMD is non-neovascularizing disease, insome instances, the growth of vessels through the pores of a substratecage could result in damage to the vessels, or disruption of the cellswithin a substrate cage. As discussed above, certain embodimentscomprise non-porous substrate cages that are permeable to nutrients fromthe bloodstream. In such embodiments, pores in the substrate cage aswell as the growth of vessels into such pores, is not possible, therebylimiting the possibility of blood vessel rupture and/or disruption ofthe cells growing on the substrate cage.

In some embodiments, certain materials may be used that are bothpermeable and porous. Selection and/or adjustment of the formulation ofselected materials (e.g., co-polymers) are used, in some embodiments, totailor the permeability and/or the porosity of the materials (and theresulting substrate).

Materials

A variety of materials may be used to fabricate the substrates disclosedherein. In some embodiments, the substrates are biodegradable while inother embodiments, the substrates are non-biodegradable. In still otherembodiments, a portion of the substrate is biodegradable while anotherportion is not. In several embodiments, the biodegradable portions ofthe substrates can be fabricated to degrade at a known rate. Suchbiodegradable materials include any suitable material that degrades orerodes over time when placed in the human or animal body. Accordingly,as the term is used herein, biodegradable material includes bioerodiblematerials.

In several biodegradable embodiments, the materials selected areoptimized to accomplish a particular rate of biodegradation. Forexample, in several embodiments employing polymers, the composition ofthe polymers is controlled to achieve a certain rate of biodegradation,and hence residency time of the substrate in vivo. By way of example, inone embodiment in which the substrate comprises a PLGA co-polymer, therate of biodegradation of the PLGA copolymer is controlled by varyingthe ratio of lactic acid to glycolic acid units in the copolymer. Insome embodiments, the rate of biodegradation is controlled to achieve aresidency to of approximately 4 weeks post-implantation. In someembodiments, the substrate degrades in about 1-3 weeks, 2-4 weeks, orlonger, including from about 4-6 weeks or several months.

In addition to the materials used to fabricate the substrate itself,several embodiments comprise an additional biodegradable layer orcoating that functions to delay the interaction between the targettissue and the cells housed within or on the substrate for a knownperiod of time. For example, a coating with a rapid rate of degradationcould be used to encapsulate the substrate and thereby protect the cellswithin or on the substrate during the implantation process and/orprevent the pores of the substrate from becoming obscured or blockedwith tissue during the implantation process. Upon completion ofimplantation, the layer would rapidly degrade and the interactionbetween the cells within or on the substrate and the target tissue wouldcommence. In contrast, in some embodiments, a more slowly degradingcoating may be used. For example, if the implantation procedure wasperformed as a surgical procedure (or in conjunction with a surgicalprocedure), post-surgery medications (e.g., anti-inflammatories and/orantibiotics) which may adversely affect the cells within or on thesubstrate may be present for an extended period of time at or near thetarget tissue. In such cases, the degradation rate of the coating couldbe tailored to prevent the exposure of the cells to the target tissueuntil a time when the harmful agent was no longer present.

Some embodiments comprise a non-biodegradable material combined with abiodegradable material, the latter which provides additional structuraland mechanical support aiding in substrate handling during cell seedingand culturing and/or during surgical insertion into a tissue (e.g., thesubretinal space). The material may also be used to add mass to thesubstrate to assist in the same and/or to assist in orientation of thesubstrate. In several embodiments, the support is in the form of columnsconnecting the top and bottom portions of a substrate cage or on thebasal portion of an inhomogeneous substrate. In other embodiments thesupport comprises an additional layer on the top or bottom of the cageor the basal surface of an inhomogeneous substrate.

The substrates may be formed of metals, polymers, plastics, orcombinations thereof. In some embodiments, the material allows thesubstrate to have sufficient elasticity, flexibility and potentialelongation to not only conform to the target anatomy during and afterimplantation, but also remain unkinked, untorn, unpunctured, and with apatent lumen during and after implantation. In several embodiments,substrate material would advantageously be processable in a practicalmanner, such as, for example, by molding, extrusion, thermoforming, andthe like, as well as by the MEMS manufacturing methods discussed below.

The purpose of surface modification, in some embodiments, is to promotecell viability and attachment. This is done by functionalizing thesurface. Towards this end, illustrative examples of suitable materialsfor the substrate include parylene polypropylene, polyimide, glass,nitinol, polyvinyl alcohol, polyvinyl pyrolidone, collagen,chemically-treated collagen, polyethersulfone (PES),poly(glycerol-sebacate) PGS, poly(styrene-isobutyl-styrene),polyurethane, ethyl vinyl acetate (EVA), polyetherether ketone (PEEK),Kynar (Polyvinylidene Fluoride; PVDF), Polytetrafluoroethylene (PTFE),Polymethylmethacrylate (PMMA), Pebax, acrylic, polyolefin,polydimethylsiloxane (PDMS) and other silicone elastomers,polypropylene, hydroxyapetite, titanium, gold, silver, platinum, othermetals and alloys, ceramics, plastics and mixtures or combinationsthereof. Additional suitable materials used to construct certainembodiments of the substrates include, but are not limited to,poly-para-xylylenes (e.g., parylene, including but not limited toparylene A, parylene AM, parylene C, ammonia treated parylene, paryleneC treated with polydopamine), poly(lactic acid) (PLA),polyethylene-vinyl acetate, poly(lactic-co-glycolic acid) (PLGA),poly(D,L-lactide), poly(D,L-lactide-co-trimethylene carbonate),collagen, heparinized collagen, denatured collagen, modified collaged(e.g., silicone with gelatin), other cell growth matrices (such asSYNTHEMAX™), poly(caprolactone), poly(glycolic acid), and/or otherpolymer, copolymers, or block co-polymers, poly(caprolactone) containingcyclic Arginine-Glycine-Asparagine, cyclic or linearArginine-Glycine-aspartic acid, blends of polycaprolactone andpolyethylene glycol (PCL-PEG), thermoplastic polyurethanes,silicone-modified polyether urethanes, poly(carbonate urethane), orpolyimide. Thermoplastic polyurethanes are polymers or copolymers whichmay comprise aliphatic polyurethanes, aromatic polyurethanes,polyurethane hydrogel-forming materials, hydrophilic polyurethanes, orcombinations thereof. Non-limiting examples include elasthane(poly(ether urethane)) such as Elasthane™ 80A, Lubrizol, Tecophilic™,Pellethane™, Carbothane™, Tecothane™, Tecoplast™, and Estane™.Silicone-modified polyether urethanes may include Carbosil™ 20 orPursil™ 20 80A, and the like. Poly(carbonate urethane) may includeBionate™ 80A or similar polymers.

Substrate Fabrication and Manipulation

Depending on the materials selected and the type of substrate to befabricated (e.g., 3-D substrate cage or asymmetric inhomogeneoussubstrate), various techniques may be used to fabricate the devices. Itshall be appreciated that the procedures listed herein are notexhaustive not meant to be interpreted as an exclusive list. Additionaltechniques known in the art, but not expressly disclosed herein may alsobe used to fabricate several embodiments of the invention. It shall alsobe appreciated that several embodiments of the substrates disclosedherein comprise combinations of materials, and therefore combinations offabrication techniques may be used.

In several embodiments, the substrate is fabricated by extrusion,drawing, injection molding, sintering, micro machining, laser machining,and/or electrical discharge machining, or any combination thereof. Insome embodiments, 3-dimensional substrates are fabricated as a singlepiece, however in several embodiments, the substrate is fabricatedmodularly. For example, in one embodiment, the top and bottom portionsof a 3-dimensional substrate cage are fabricated independently of oneanother. Such an approach is advantageous in the production ofsubstrates that differ from one another in one or more aspects (e.g., afirst substrate has porous apical and basal surfaces and a second has anon-porous apical surface), but retain the same overall dimension. Insuch embodiments, the desired modular pieces may be selected and thenassembled into a complete 3-dimensional substrate cage. After thedesired modular pieces have been selected, they are aligned and sealedto create a 3-dimensional substrate cage with the desired dimension andcharacteristics. In some embodiments, a 3-dimensional substrate cagefabricated from a single piece is also sealed. For example, in oneembodiment, the top and bottom portions are formed as a single flatpiece and then one portion is folded over the other and the resultant3-dimensional substrate cage is sealed. Sealing may be accomplished withheat welding, annealing, biocompatible adhesives or epoxies, and thelike. Some embodiments optionally include a shell or frame comprisingadditional material that does not function as a cell growth surface. Insome embodiments, the shell or frame provides a user one or more placesto grip and or manipulate the substrate without damaging the cell growthsurface and/or the cells growing on said surface.

While 3-dimensional substrate cages that function to foster interactionbetween the cells and the target tissue while retaining cells in thecage post-implantation are preferred in several embodiments, asdiscussed above, several embodiments comprise a substrate material(e.g., a biodegradable material) having at least one homogeneous cellgrowth surface and additional features for structural support. Some suchembodiments are advantageous in that their production may be simplifiedas compared to a 3-dimensional substrate cage. Moreover, in severalembodiments, substrates with one homogeneous apical cell growth surfaceare suitable for positioning in a target site in a manner that stillretains the cells on the homogeneous (apical) surface of the substrateand facilitates the therapeutic interaction with the target tissue. Inother words, the lack of a 3-dimensional or cage-like structure does notreduce the therapeutic efficacy of substrates as disclosed herein.

In several embodiments, manufacturing of a plurality of substrates (orthe modular portions thereof) is accomplished simultaneously. In someembodiments, the cells to be used for therapeutic effect are grown onthe material that comprises the substrate (or a portion thereof) priorto the final fabrication of the substrate. In some embodiments, thesubstrates (or modular portions thereof) are effectively used as an invitro culture substrate for the cells and at an appropriate time, areprocessed through the final phases of fabrication (if any are needed,depending on the embodiment) and ready for in vivo implantation with thecells pre-loaded in or on the substrate. For example, in one embodiment,a large sheet of polymer material is used as a substrate to grow aplurality of cells to be delivered in controlled laboratory conditions.When the cells are determined to be in an optimal state for delivery(e.g., a certain phase of cell cycle or a certain population density), aplurality of individual substrates are removed from the polymer sheet,sealed and ready to be implanted.

Several embodiments with homogeneous apical surfaces are particularlyamenable to seeding and growth of cells simultaneously on a plurality ofsubstrates, which are joined during the culturing process and areseparated into individual substrates prior to insertion or implantation.For example, as shown generally in FIGS. 18A-18C, multiple substratescan be fabricated from one contiguous piece of the chosen material(e.g., a biodegradable polymer; hereafter referred to as the frame). Asshown in FIG. 18A, a single circular frame is capable of providingmechanical support for multiple individual implantable substrates. Inseveral embodiments, each substrate is attached to the larger frame viaan extension (e.g., a handle; see generally FIGS. 18B and 18C).Dimensions of the substrates and handles are described in greater detailabove. In several embodiments the handle of the substrate is free ofcellular growth, allowing for manipulation and loading onto a surgicaltool without disrupting monolayer integrity.

In several embodiments, the frame is dimensioned to maximize the surfacearea available for growth of cells seeded onto the substrate. Forexample, the frame may optionally be made in a rectangular shape and becomprised of a plurality of rectangular substrates. In one embodimentthe rectangular substrates plus frame taken together maximize polymer toexposed culture dish surface area ratio by employing an interdigitating“comb-like” structure (see e.g. FIG. 18D). It shall be appreciated thatthis figure is merely representative and a greater or lesser number ofimplants can be used, depending on the embodiment. In severalembodiments a circular frame is preferred, as such frames can bedimensioned to fit within a standard cell culture dish (e.g., a 10 cmdish). In such embodiments, the close juxtaposition of the edges of theframe with the walls of the culture dish reduce flow and turbulence ofthe culture media over the cell growth surface, which can disrupt theintegrity of certain growing cell populations. In some embodiments,larger or smaller diameters of substrate frame are used (e.g., diametersbetween about 5 cm to about 10 cm, from about 6 cm to about 9 cm, fromabout 7 cm to about 8 cm, and overlapping ranges thereof (frames mayalso be sized to fit in any variety of culture vessel as describedherein).

As discussed herein, the plurality of individual substrates can befabricated from a single, larger substrate frame using photolithographictechniques, injection molding, and the like.

Also as discussed herein, the individual substrates may be, withoutlimitation, circular, oblong, or any shape customized to a specific toindividual patient pathology (see e.g., FIGS. 5-8). Customization ofsubstrates is described in more detail below, by can be determined byelectrophysiological testing (e.g. mfERG), psychophysical testing (e.g.kinetic or static microperimetery), or various ocular imaging modalities(i.e. spectral-domain optical coherence tomography (SD-OCT), fundusphotography, fundus autofluoresence (FAF), or confocal scanning laserophthalmoscopy (cSLO).

In several embodiments, the substrate frame is not designed tonecessarily optimize or maximize a contiguous cell-growth surface, butrather is configured to allow selection and removal of a singlesubstrate without disturbing other substrates on which cells are stillgrowing. As shown generally in FIG. 22A, in some embodiments, aplurality of substrates 10′ are fabricated on a single circular frame130 positioned in a manner which allows the handle 30′ of the substrateto be accessed by a manipulation tool (e.g., forceps). Once fabricated,a single circular substrate can then be seeded with cells (e.g., H9hESC-RPE) and cultured until such time that the cells have reached anoptimal growth state (e.g., a confluent monolayer over the substrate).Individual oblong substrates are then cut from the larger frame using asterilized and autoclaved pair of scissors or a custom designedholding/cutting tool. In several embodiments, each substrate comprisesan identifier 155′ to aid the surgeon in orienting the substrate. Insome embodiments, the identifier comprises a visual or chemicalindicator (e.g., a fluorescent dye that can be visualized). In someembodiments, a MEMS reporting system is employed, and can report theviability of the cells or other local environmental conditions aroundthe substrate. In several embodiments, a metal boundary or point isembedded into the substrate. Suitable metals include, but are notlimited to nitinol, titanium, gold, silver, platinum, other metals andalloys, foils made from the same, and the like. In some embodiments, thesubstrates are doped with a fluorophore for imaging the substrate. Insuch embodiments, the loss or migration of cells from the substratecould be identified post-implantation or post-operatively, thusproviding a means to assess the quality of the implantation procedureand to determine whether an alternate or additional substrate should beimplanted. In some embodiments, the substrates further comprise a barcode or other unique identifier for quality control lot/batchinformation and inventory purposes.

A non-limiting example of a custom holding/cutting tool 140 is shown inFIG. 22B. The cutting tool in fact functions in multiple additionalways, beyond simply enabling the cutting of an individual substrate fromthe frame. For example, in several embodiments the tool functions as aweight that holds the substrate frame in position within a culturevessel. In several embodiments, the holder/cutter device is reversiblyattached to the underside of a tissue culture dish. In severalembodiments, the holder/cutter device is integrated into the undersideof a custom tissue culture dish. In such embodiments, the holder/cutteris advantageously pre-sterilized. In some embodiments, this isparticularly advantageous, as some cell types require a greater mediavolume, which increases the flow of media throughout the vessel duringnormal handling. Such fluid flow could disrupt the integrity of growingcell populations, thereby increasing the overall time to prepare ansubstrate for implantation and/or adversely affecting the viability ofthe cells on the substrate. Further, the tool allows for the consistentand repeatable cutting of individual substrates.

Moreover, the tool, as it has a plurality of contact points 150 with theframe, prevents the growth of cells on a portion of the handle of thesubstrate. This cell-free portion is the portion which is grasped bymanipulating tools (e.g., forceps) that are used in some embodiments. Insuch embodiments, the use of forceps does not disrupt the cells growingon the substrate growth surface, thereby maintaining the integrity andviability of the cells during the transition from the culture vessel tothe target site of a subject. Each contact point also comprises a slotor aperture 151 that is dimensioned to allow a cutting device (e.g., ascalpel or custom designed sterilizable blade) to be inserted throughthe contact point and cut the underlying substrate handle, therebyfreeing the individual substrate from the frame. The associated cuttingpoint on the substrate frame is shown as 151′ in FIG. 22A.

In some embodiments, substrates that are cut from the frame aremanipulated and implanted with a specialized tool, which is described inmore detail below.

Arrangements comprising a plurality of substrates within a frame areadvantageous in some embodiments, because of the plurality ofsubstrates, a certain number substrates may be used for release testingof the lot (e.g., assays testing genotype, phenotype, and function),which consists of one or multiple circular frames, while another portionof the substrates can be retained for implantation in a subject.Moreover, such a layout enables evaluation of each of the substrates inthe frame for identification of the substrate that best suits aparticular implantation procedure (e.g., has appropriate cells numbers,cell density, etc.) and subsequent selection of that substrate, withoutperturbation of the other substrates in the frame.

Fabrication of a plurality of substrates with cells pre-grown on thematerial provides certain additional advantages in some embodiments. Inone embodiment, the possibility of contamination of the cells isminimized because the cells (and the material they are grown on) arealready in sterile culture conditions and manipulation prior toimplantation is limited.

Moreover, in some embodiments, growth of cells on a plurality ofsubstrates allows the selection of the most healthy and viable cellpopulations prior to implantation. Additionally, those cells that arenot optimal at the time of evaluation need not be discarded, but can becultured for a longer period of time and/or under different conditions,until such time as they are optimal for implantation. In someembodiments a single point of connection between each substrate and thelarger frame aids in simple cutting or dislocation of the substrateprior to surgery, further minimizing possible contamination of or damageto cells compared with similar designs requiring the full stamping-outor cutting of the substrate along it's entire perimeter (which maycompromise the health and/or integrity of one or more cells on thesubstrate periphery). Furthermore, scale-up in manufacturing of aplurality of substrates with cells pre-growing is easily accomplished.

In addition to the methods used in the fabrication of the substrate, insome embodiments, additional processes are employed to further adapt thesubstrate, or the materials the substrate is fabricated from, to theparticular cells to be used or target tissue. For example, in oneembodiment, a polymer substrate, such as parylene, is used to fabricatethe substrate. In one embodiment, the material surface that will formthe cell substrate of the fabricated substrate is hydrophilicallymodified using oxygen treatment. This hydrophilic treatment generates anideal surface for certain cell types to grow on.

In some embodiments, oxygen plasma treatment is performed using areactive ion etch (ME). The etch is performed for two minutes at a powerof 100 W and a maintained O₂ chamber pressure of 200 mTorr. In severalembodiments, surface modification of parylene-c allows for the surfaceto remain hydrophilic for an extended period of time compared with othercommonly used biocompatible polymers. O₂ plasma treatment will allow, insome embodiments, for maximal density of seeded cells, thus increasingthe effective dose of the therapeutic to the targeted area.

For example, retinal pigmented epithelial cells (RPE) grow particularlywell on a hydrophilic surface, as the RPE cells are polarized and thusorient themselves with respect to the hydrophilic surface. In severalembodiments, the substrate enhances the ability of cells seeded thereonto polarize, thus reducing the likelihood of shedding or migration offthe substrate during the delivery process.

In several embodiments, one or more surfaces of the substrate arealtered to enhance handling and/or visualization of the substrate. Forexample, the outer surfaces of the substrate may be etched in order toroughen the surface. The rougher surface, in some embodiments, providesa surface which diffracts light to a greater degree than a smoothsubstrate surface, which reflects light. In some embodiments, thisdiffraction of light allows a user to visualize the substrate and/ortissues underneath or distal to the substrate more clearly.

MEMS Features

As discussed above, in several embodiments, the substrate are MEMSdevices and/or incorporate MEMS technology. In several embodiments, thesubstrate comprises a central unit for data processing, one or moremicrosensors that evaluate the cellular environment within the deviceand or surrounding the target tissue. In several embodiments, thesubstrate further comprises a reporting unit that indicates certaininformation about the environment surrounding the substrate or the cellswithin the substrate. In some embodiments, the substrate reports on theviability or metabolic condition of the cells within the substrate. Insome embodiments electrodes can be used to report the electricalimpedance value at the device-tissue interface, and quantify proximityand/or mechanical force and pressure. These electrodes are also used, insome embodiments, to measure oxygen concentration and/or to measureblood flow. In some embodiments, the substrate reports on the degree ofinteraction between the cells within the substrate and those of thetarget tissue. For example, in certain ocular embodiments, the devicereports to a user information regarding the degree of physicalinteraction taking place between the RPE cells within the device andtarget photoreceptor cells. In some embodiments, Fast CV is used tomonitor the basal concentrations of electroactive species (as discussedabove) to assess the health of the cells and tissues. This isadvantageous in some embodiments, because photo-oxidative stress is aknown cause of AMD and the device would be implanted close enough to thechoroid to measure rate of blood flow. Moreover, in several embodiments,the device allows for assessment of underlying cellular morphology andhealth through the use of advanced imaging tools (e.g., spectral-domainoptical coherence tomography (SD-OCT), fluorescein angiography (FA),fundus photography, SD-OCT supplemented with adaptive optics(AO-SD-OCT). In one embodiment, the boundary between RPE cells withinthe device and the endogenous photoreceptors (thePR-RPE-choriocapillaris boundary) is visible using long-wavelengthSD-OCT imaging, thereby enabling adequate assessment of the ability ofthe cells to restore function.

In several embodiments, the substrate is configured to electrochemicallydetect electroactive species (e.g., neurotransmitter concentration, suchas noradrenalin (norepinephrine), adrenaline, serotonin and dopamine).In some embodiments, fast cyclic voltammetry, proximity to tissuemeasurement using impedance, or other similar electrochemical detectionmethods are used to allow the device to report on the presence and/orconcentration of electroactive species.

In several embodiments, the features of the substrate described hereinfacilitate the use of imaging techniques. In particular, ocular imagingtechniques are used in some embodiments. In some ocular-directedembodiments, for example, the features of the device allow theassessment of the health of endogenous RPE and PR. Optical coherencetomography (OCT) is used in some embodiments and allows non-invasiveimaging (e.g., without injection of dyes or radioactive labels) with ahigh degree of resolution. Such techniques allow for the imaging ofabout 10-100 cells in an intact eye. Moreover, advanced imagingtechniques allow for the assessment of blood flow dynamics at thecapillary level that supports RPE cells (choriocapillaris), and also theassessment of bleaching and rejuvenation of photoreceptor visualpigments; both of these are important metrics of the functionality ofthe RPE-PR complex.

Advantageously, many of the materials used in traditional MEMS devicesare those described herein as being suitable for fabrication of thesubstrate. For example, the MEMS components of the device may befabricated from, for example, polymers, silicon, or various metals. Inthose embodiments in which polymer-based MEMS devices are fabricated,processes such as injection molding, embossing or stereolithography maybe used. In those embodiments in which silicone-based MEMS devices arefabricated, procedures such as deposition of material layers, patterningof the layers by photolithography, and then etching to produce therequired shapes. In those embodiments in which metal-based MEMS devicesare fabricated, procedures such as electroplating, evaporation, and/orsputtering may be used. Other processes such as molding and plating, wetetching or dry etching, electro discharge machining (EDM), and othersimilar processes known in the art are used in several embodiments.

In several embodiments, MEMS features are used to anchor a device to atarget tissue. In some embodiments, MEMS latches or clamps are used tograsp the surface of a target tissue.

Custom Fabrication

In several embodiments, substrates are custom fabricated depending onthe disease to be treated and the particular characteristics or symptomsof the individual afflicted with the disease. In several embodiments, aphysician will make a determination regarding the distribution ofdamaged or diseased cells in particular patient. As a result, acustomized substrate is generated that places the substrate and thecells associated with the substrate in the regions correlating to thedamaged or disease cells.

For example, in ocular applications, the visual field of a patient canbe determined by any appropriate known diagnostic techniques, such asGoldmann kinetic perimetry (see, e.g., FIG. 5). Thereafter (or in placeof), local visual function can be determined by any appropriate knowntechnique, to generate a multi-focus electroretinogram (see, e.g., FIG.6). With this data, a custom substrate can be fabricated that placescells in juxtaposition to the area of lost or diminished visualfunction, that area corresponding to dead or functionally damagedphotoreceptors.

In several embodiments, an injection mold is formed based on thedetermination of an individual's visual field/visual function (see e.g.,FIG. 7). In some embodiments, the mold comprises plastic, aluminum, orother easily workable (e.g., machinable) materials. In some embodiments,the mold can be stamped or shapes from elastomeric materials in aphotographic process flow. In several embodiments, the materials arespin coated onto larger discs of material prior to stamping, therebygenerating more uniform thicknesses in the resultant stamped substrate.In some embodiments, the mold form corresponds to the apical or basalsurface of the substrate. The material of choice for the substrate, forexample parylene (or other suitable material described herein) is shapedto the mold. In one embodiment, a solution of parylene polymer moleculesis deposited into the mold and UV cured. The apical and basal surfacesare thereafter aligned and sealed, as described herein. In severalembodiments, a metal trace is deposited on the apical and basal surfacesof the substrate (e.g. around the perimeter) to enhance alignment andsealing of the two portions of the substrate (see element 90 in FIG. 8).In some embodiments, a platinum trace is deposited on the substratehalves by electron beam evaporation followed by electrochemical platinumdeposition. In some embodiments, an Indian-tin oxide is deposited withsealing of the halves accomplished by flip-chip bonding. In severalembodiments, MEMS latches are used to attached the various substratecomponents to one another. Other metal traces and bonding techniquesknown in the art are used in other embodiments. Not only does this metaltrace provide enhanced alignment of the halves of the substrate, it alsooptionally provides additional thickness (depending on the amount ofmetal deposited) and adds structural support to the substrate (e.g., toprevent folding or crimping during the implantation process).

In several embodiments, the cells housed in such a substrate cage forocular therapy are RPE cells. It shall be appreciated that RPE cells arealso used in other non-cage embodiments. Substrates in accordance withseveral embodiments described herein are particularly advantageous foruse in ocular applications, as the function of damaged photoreceptorscan be supported (e.g., function is regained or further loss of functionis prevented and/or minimized) by a physical interaction between the RPEcells retained within the substrate and the photoreceptors; RPEengraftment is not required.

In several embodiments, the RPE stem cells are modified to secreteneurotrophic factors that further support survival of photoreceptorcells. In certain such embodiments, genes encoding one or moreneurotrophic factors are cloned into the RPE cells prior totransplantation such that there is significant and enhanced protectionafforded to the photoreceptor neurons. Examples of such possible factorsinclude PEDF, CNTF, BDNF. Over expression of other genes may allow forenhanced function of RPE. Examples include: over expression of surfaceintegrins (which have been shown to increase RPE adhesion to BM),melanin (ethnic groups with increased melanin production are at lowerrisk for AMD), or receptors required for phagocytosis of shed discs(e.g. CD36, MerTK). In some embodiments, over expression of receptorsrequired for phagocytosis is advantageous because at the time oftherapeutic intervention there is likely to be an accumulation oflipofiscin and metabolic waste products that would require removal.

In several embodiments, the substrates and cells housed therein areemployed in the treatment of age-related AMD, and in several preferredembodiments dry AMD. Other approaches for treatment of dry AMD includemacular translocation and autologous RPE transplantation. Both arecomplex surgical procedures requiring general anesthesia and both areassociated with high rates of retinal detachment. Moreover, there are noFDA-approved, effective pharmacologic therapies for dry AMD.Multivitamins only slow the progression of early AMD. With regard toother cell therapy approaches, differentiation of hESC or retinalprogenitors into photorecptors is complex, as implanted photorecptorswould have to integrate and form synapses with the host tissue. Incontrast, the substrates disclosed herein allow the support of existingphotoreceptors through the interaction between RPE cells within thesubstrate without the need for engraftment and/or synapse formation.Moreover, several embodiments of substrates disclosed herein promote theformation of a monolayer of cells within the substrate, which isadvantageous as compared to a cell suspension. In some embodiments, amonolayer of cells on a substrate more closely mimics the structure ofBruch's membrane, which cannot, in AMD, provide a substrate for theattachment and further differentiation of injected cell suspensions.Thus, some embodiments of the substrates provide a synthetic replacementfor the tissues damaged in AMD, rather than attempting to deliver cellsthat may not have an optimal tissue to attach to in vivo.

Cell Growth

In several embodiments, the substrate cages (and/or inhomogeneoussubstrates) are fabricated in a manner that provides an ideal surfaceand environment for the growth, survival, and function of cells. Asdiscussed above, various characteristics of the substrates may bealtered in order to surgical aspects of the substrate. To reiterate, theshape, thickness, pore diameter, and pore density are varied, in certainembodiments to optimally allow the housed cells to thrive and produceinteractions with the target tissue, while being retained within (or on)the substrate. Thus, in some embodiments, the substrate confers thetherapeutic benefit of the housed cells onto the target cells withoutthe engraftment of the cells housed within the substrate. For example,in one embodiment, an epithelial layer of cells within the substrate(e.g., RPE) that are differentiated from hESC treat neurologicaldegeneration (e.g., photoreceptor degeneration), without the need forsynapse formation or neural integration. Moreover, these characteristicscan be optimized to prevent migration of the cells out of (or off) thesubstrate and into the target tissue. Further, these characteristics maybe varied in order to provide sufficient structure and resilience forthe substrate to be implanted in vivo without damaging the substrate orthe cells housed in the substrate.

As discussed above, the cell growth surface may also be altered (eitherbefore or after fabrication is complete) to optimize the surface for aparticular cell type. In several embodiments, the cell surface istreated with oxygen to generate a hydrophilic cell growth surface on thesubstrate. In some embodiments, the oxygen treatment further comprisesan additional compound, such as a matrigel, to support growth ofpolarized cells (e.g., RPE). In several embodiments, such matrigelcompounds are xeno-free. Also as discussed above, in certainembodiments, the pore diameter may be adjusted depending on the celltype to be housed in the substrate.

In several embodiments, immunosuppressive agents are also delivered inorder to avoid rejection of allogeneic cells introduced by thesubstrate. In some embodiments the substrate may be used to providetargeted and released delivery of these agents or drugs such ascorticosteroids (e.g. prednisone), methotrexate, cyclosporine,antimetabolites, T-cell inhibitors, and alkylating agents. Theadministration of such immunosuppressive agents is used to avoidrejection of grafts or transplants or in treatment of autoimmunedisorders. However, high-doses may be required, and oral administration,or even more localized and targeted delivery via injection, can bedangerous due to the increased risk of infection. In severalembodiments, introduction of these agents via controlled and targetedrelease from substrates disclosed herein increases the likelihood ofallogeneic graft acceptance, and minimize the risk of infection causedby immune suppression.

Other drugs that have been used clinical trials for treatment to AMD canbe released by the substrate in a similar controlled and targeted mannerby embedding in a bio-degradable portion or in the interconnect caseitself. Examples include lipid-lowering statins, retinoids (e.g.Acitretin, Alitretinoin, Bexarotene, Etretinate Fenretinide,Isotretinoin, Tazarotene, Tretinoin), and anti-VEGF drugs.

As discussed above, in several embodiments a second substrate isannealed to the primary substrate. For example, in some embodiments, abiodegradable substrate is employed. In one embodiment, a biodegradablepolycaprolactone (PCL) material compounded polyethylene glycol (PEG) isused. The polymerizing PCL-PEG material is spin-coated onto glasscoverslips to make thin films of a desired thickness. Upon treatmentwith aqueous buffer or media, the water soluble PEG is washed away andcreates pores in the film. As discussed above, biodegradable embodimentsmay be customized, and in one embodiment, the ratio of the PCL to PEGaffects the degradation rate of the substrate. As the PEG is washed awayto form pores, the pores increase the surface area of the polymerexposed to water and therefore increase the degradation rate. Throughthis approach both the porosity of the films as well as the degradationrate can be manipulated based on the amount of PEG added to the polymerblend. In some embodiments, polymers with cyclic amino acids(Arg-Gly-Asp; cRGD) are annealed to the polymer film, thereby providinga polymer surface that promotes cellular adhesion and growth bymimicking the extracellular matrix protein, fibronectin.

Cell Loading

In several embodiments, cells to be used in or on the deliveredsubstrate are cultured on the substrate prior to final fabrication ofthe substrate. As discussed above, this provides several advantages withrespect to minimizing the risk of contamination or damage to the cellsor the substrate. However, in several embodiments, the cells aredeployed into or on a completely fabricated and sterilized substrate.Substrates as described herein may be sterilized by gamma irradiation,ethylene oxide, autoclaving, UV sterilization, or other known procedureswithout degradation or damage. Such cell deployment is carried out understerile cell culture or sterile surgical suite conditions. Suchembodiments have the advantage, among others, that optimally healthy androbust cells can be selected and deposited into the substrate just priorto implantation. For example, in several embodiments, cells are loadedinto a 3-D substrate cage by a surgeon while in the operating room. Thismethodology is advantageous because a surgeon could select from severalvarieties of substrates, depending on patient characteristics or otherparameters, and then load optimally healthy cells into the substrate. Insome embodiments, a vacuum pressure device functions to assist thesurgeon on holding and filling the substrate cage with cells. In severalembodiments, this is advantageous because the vacuum pressure that holdsthe substrate cage also allows the volume of cell delivery fluid (thatis potentially larger than the volume of cells) to be removed from thesubstrate cage through the biological vias. In some embodiments, thesubstrate is loaded into a surgical introducer and then loaded withcells. In some embodiments, the substrate is loaded with cells, thenplaced in the surgical introducer. In several embodiments usingasymmetrical inhomogeneous substrates, cells are pre-seeded and stablygrowing on the apical surface of the substrate, before selection andsubsequent implantation of the implant by a surgeon.

With reference to FIGS. 2A-2B, and FIG. 9, some embodiments of thesubstrate comprise a cell retention feature 70. Such a feature functionsto provide a one-way passage for cells into the lumen 50 of thesubstrate cage. In several embodiments, the retention feature is customshaped to provide an interlock with a pipette (or other device) 100 thatdelivers the cells into the lumen of the substrate cage. In severalembodiments a resealable or “self-healing” membrane is used such that apipette or needle can puncture through the membrane into the lumen fordelivery of cells, but upon withdrawal of the pipette or needle, leavesno open orifice for cells to escape from. In some embodiments aviscoelastic is used. In some embodiments a biocompatible adhesive isused. In still other embodiments a one-way valve is used. Such a valvemay comprises two or more flaps which open into the lumen uponadvancement of a delivery pipette, which allows for the deposition ofcells into the lumen. Upon removal of the pipette, the flaps return totheir closed position, thereby retaining the deposited cells within thelumen. In some embodiments, the one way valve is formed such that aliquid tight seal is created to prevent backflow of cells, while inother embodiments, a fluid-tight seal is not formed.

Cell Types

Given the wide variety of diseases that induce cell damage or celldeath, a wide variety of cell types can be housed within substratesdescribed herein to achieve therapeutic effects. In some embodiments,cultured cells are used. In several embodiments, banked cells are used.In some embodiments, the cultured cells comprise stem cells. Stem cellsare pluripotent cells capable of differentiating into a variety ofdifferent cell types. In some embodiments, embryonic stem cells areused, while in other embodiments, adult stem cells are used. In severalembodiments, the embryonic stem cells are human embryonic stem cells.Embryonic stem cells, which are typically derived from an early stageembryo, have the potential to develop into any type of cell in the body.In some embodiments, H1, H7, H9, SHEF-1, or other similar FDA-approvedstem cell lines are used. Adult stem cells are typically multipotent andcan develop into a more limited number of cell types, typically thosethat are related to the tissue type from which the cells were isolated.In some embodiments, the stem cells are allogeneic to the recipient(e.g., as is the case with embryonic stem cells). In some embodiments,the stem cells are autologous to the recipient. In other embodiments,syngeneic cells are used, while in still other embodiments, xenogeneiccells are used. In some embodiments, freshly isolated cells are culturedand deployed into or onto the substrate for implantation into arecipient individual. In other embodiments, cryopreserved cells areused. In some embodiments, induced pluripotent stem cells are used.

In several embodiments, stem cells are isolated (or thawed) and culturedunder standard sterile in vitro culture conditions. In some embodiments,the cells are cultured in standard tissue culture media known and usedfor the growth of stem cells. In other embodiments, a xeno-free culturemedia is used. In some embodiments, culture media is supplemented withone or more growth factors in order to support the viability of thecells and/or induce differentiation into a particular cell type. In someembodiments, growth factors such as are included in serum used tosupplement the growth media. In some embodiments, specific factors areadded, such as, for example, fibroblast growth factor, transforminggrowth factor (e.g., TGFβ1), insulin-like growth factor, e.g., IGF-1).Cells may be grown, passaged, and continually cultured until such timethat the cells have optimal characteristics for deployment into thesubstrates described herein. At that point, the cells (if suspensioncells) may simply be collected, adjusted to a desired cell density, anddeployed (e.g., injected) into the substrate. Cells that are grown on asurface may be enzymatically digested (e.g., trypsinized) ormechanically detached from the surface, adjusted to a desired celldensity, and deployed (e.g., injected) into the substrate.

Due to the features and advantages of several embodiments of thesubstrates described herein, in several embodiments, cell numbers thatare deployed into or onto the substrate are relatively low. In severalembodiments, the number of cells delivered ranges from about 1.0×10³ to1.0×10⁸. In several embodiments cell numbers deployed into or onto thesubstrate range from about 1.0×10⁴ to about 1.0×10⁷, from about 1.0×10⁵to about 1.0×10⁶, and overlapping ranges thereof. In one embodiment,about 1.5×10⁵ cells are used. In other embodiments, greater or lessernumbers of cells are used.

As discussed above, cells from a variety of tissues may be used. Inseveral embodiments, ocular cells are used to treat ocular diseasesincluding, but not limited to age related macular degeneration (wet ordry), diabetic macular edema, idiopathic choroidal neovascularization,or high myopia macular degeneration. In some ocular embodiments, RPEcells are used. In several embodiments, cardiac stem cells are used totreat cardiovascular disorders such as myocardial infarction, ischemiccardiac tissue damage, congestive heart failure, aneurysm,atherosclerosis-induced events, cerebrovascular accident (stroke), andcoronary artery disease. In several embodiments, liver stem cells areused to treat liver disease such as hepatitis, cirrhosis, cancer, andthe like. Diseases in other tissues, such as the kidney, lung, pancreas,intestine, and neural tissues, among others, may be treated with themethods and devices disclosed herein. In some embodiments, harvestedbone marrow stem cells may be used to repopulate hematopoietic cellsthat are reduced due to leukemias, cancers, or therapies that reduceblood cell counts.

Delivery Methods

Substrates in accordance with embodiments described herein may bedelivered by various methods depending on the target tissue. Substratesmay be delivered during an open surgical process. For example, duringocular surgery a substrate may be delivered to a region of the eye. Inseveral embodiments, substrates are implanted in a specific deliveryprocedure. In some ocular embodiments, the substrates are delivered tothe sub-retinal space. In some embodiments, an ab-interno procedure isused. In other embodiments, an ab-externo procedure is used. In someembodiments, a pars plana surgical approach is used for implantation. Inother embodiments a trans-scleral approach is used for implantation. Inseveral embodiments, substrates are attached (e.g., sutured, adhered) toa surface. In several embodiments, substrates are deliveredendoscopically, via catheter-based methods, intravascularly,intramuscularly, stereotactically (e.g., for delivery of thesubstrate/cells to the brain or other neural tissue) or by other meansknown in the art for a particular target tissue. Depending on thesubstrate design and the target tissue, customized surgical tools areused to make the delivery of the substrates less traumatic, faster, orotherwise less risky or more beneficial to the subject.

In several embodiments, the surgical approach for ocular implantation isa pars plana approach. In some species, the pars plana liesapproximately 3.5-4 mm away from cornea. In several embodiments, thesubstrate is delivered parallel to the posterior eye wall.

EXAMPLES

Examples provided below are intended to be non-limiting embodiments ofthe invention.

Example 1 Determination of Pore Diameter that Retains RPE Cells

In the eye, a healthy Bruch's membrane functions as a molecular sievethat regulates the exchange of nutrients and metabolic wastes betweenthe retina and the choroid. Based on the sub-retinal location of certainocular-directed substrates disclosed herein, the porosity of thesubstrate would ideally simulate these functions of a healthy Bruch'smembrane.

To investigate the pore diameter range that would allow for function,cell migration assays were performed. Circular parylene discs weremanufactured with varying pore diameters (1, 2, 3, and 5 um) and placedon top of Transwell polyester (PET) cell culture inserts with 8 μmpores. Inserts were placed into 24-well culture plates. RPE cells wereseeded on the parylene discs with serum free medium inside the PET cellculture inserts and medium containing 20 μg/ml recombinant human PDGF(as a chemoattractant) outside the inserts. After overnight culture, thePET insert membranes and the 24-well culture plates that held theinserts were both stained with hematoxylin. Results indicated that RPEcell bodies could migrate through 2, 3 and 5 μm diameter pores, but notthrough 1 μm pores. Thus it was concluded that pore sizes ranging fromabout 0.5 to about 1.5 μm are optimal for certain RPE-containingsubstrates.

Example 2 Biodegradable Polymer Substrates

As discussed above, parylene substrates are used in several embodiments.Biodegradable polycaprolactone (PCL) material compounded withpolyethylene glycol (PEG) is used to test the formation of pores insubstrates. During polymerization, the PCL-PEG material is spin-coatedonto glass coverslips to make thin films of a desired thickness, asdiscussed above. Cyclic amino acids (Arg-Gly-Asp; cRGD) covalently boundto a PEG-PCL copolymer is annealed onto the surface of the films. Upontreatment with aqueous buffer or media, the water soluble PEG is washedaway and creates pores in the film. The ratio of the PCL-PEG affects thedegradation rate of the substrate. These pores increase the surface areaof the polymer exposed to water and therefore increase the degradationrate. Through this approach both the porosity of the films as well asthe degradation rate may be manipulated based on the amount of PEG addedto the polymer blend.

Example 3 In Vitro Analyses of hESC-RPE

The present example demonstrates that hESC colonies grown tosuperconfluency, in the absence of FGF2, on mouse or human feeder layersor on matrigel, routinely produce discrete pigmented foci, which grow insize. These pigmented foci can be excised and expanded to produce highlydifferentiated monolayers that are similar to cultured fetal RPE oradult human cultured RPE. See FIG. 11. Cells can be produced inquantities required for clinical use at 99% purity, as determined bymicroscopic analysis of pigmented cells as well as quantitative PCR fornanog and Oct-4, markers of possible contaminating undifferentiatedhESC. Monolayer cultures are phenotypically stable after prolongedculture (11 months) without passage. Furthermore phenotype and normalkaryotype can be maintained for up to at least 4 passages. Based on cellnumbers in culture, some embodiments employ about 1.5×10⁵ cells on a 5mm diameter substrate are used for each treated eye.

Multiple RPE lines derived from different hESC lines have beencharacterized with respect to global RNA expression pattern, andquantitative analysis of key RPE marker mRNAs and proteins. HumanESC-RPE are remarkably similar to native human fetal RPE in theirexpression patterns (correlation coefficient of 0.97 between hESC-RPEand cultured fetal human RPE). Quantitative RT-PCR showed similar levelsof RPE-specific transcripts important in gene regulation (Mit-F, OTX-2,Rax, Six-3), pigment synthesis (Tyr, Tryp-1, Tryp-2, Silver), retinolproduction (CRALBP, RPE65), tight junction formation (ZO-1, Claudin-3),and other key RPE markers (Bestrophin, Emmprin, Transthyretin, PigmentEpithelium Derived Factor (PEDF). hESC-RPE express proteins associatedwith these transcripts, as well as other crucial RPE markers (integrins,laminins, fibronectin, apoE, fibulin-5, pMe117. The expression of RPE65by these cells is of particular significance, as deficiencies in thisprotein leads to blindness.

Like native RPE, hESC-RPE monolayers are polarized with apicalmicrovilli, basal nuclei and endfeet, and lateral tight junctions, asdetected by electron microscopic analysis and measurement oftrans-epithelial resistance (TER). They carry out targeted membranetrafficking with apical localization of Na/K ATPase, integrinalphavbeta5 expression, apical secretion of PEDF, and basal secretion ofcollagen IV and VEGF. In addition, the apical surface of HESCRPEincludes 1-2 cilia per cell that express a-acetylated tubulin, with a9+0 structure typical of human fetal and early postnatal rodent RPE.Polarized sheets of hESC-RPE express tight junction proteins (occludin,claudin and Z01) localized to cell-cell contact regions typical ofmature RPE cells, and generate TER typical of native RPE in situ. SeeFIG. 12A.

To show that hESC-RPE cells are capable of efficient phagocytosis ofphotoreceptor outer segments (a process which occurs on a diurnal cyclein vivo and is required for vision), fluorescently tagged photoreceptorrod outer segments (ROS) were added to sheets of hESC-RPE, fetal RPE, orH2S7 fibroblasts (negative control) in vitro and binding/phagocytosiswas quantified. See FIG. 12B. hESC-RPE are quantitatively similar tofetal RPE in their phagocytic ability, and that activity was blocked byantibodies to alphavbeta5 integrin or MerTK receptors. Additionally,primary human retina (obtained from macular translocation surgery) wereplaced onto sheets of hESC-RPE, and phagocytosis of human photoreceptorouter segments was detected.

Additional function analyses are performed in some experiments. Forexample, in several embodiments isomerization is ofall-trans-retinaldehyde to 11-cis-retinaldehyde is evaluated. The is afunction performed by the RPE in situ which is important to the visualcycle and maintenance of vision. The ability of hESC-RPE to perform thisenzymatic isomerization will be monitored by HPLC analysis of theconversion of all-trans-retinyl palmitate to the 11-cis form in aqueouscell homogenates. Another important function of RPE cells in situ is thephagocytic clearance of shed photoreceptor outer segment membrane.Phagocytosis is monitored using bovine rod photoreceptor outer segmentslabeled with SNAF1-2 (Molecular Probes) so as to allow differentiationbetween surface-bound and internalized outer segments. Pigmentepithelial derived factor (PEDF) is a major secretory product of RPEcells in situ. It acts as an inhibitor of angiogenesis and has potentneuroprotective properties. Its secretion by cultured human fetal RPEand hESC-RPE is characteristic of establishment of a polarizedepithelial monolayer with high trans epithelial resistance in severalembodiments, therefore, the presence of PEDF in conditioned medium fromhESC-RPE cells is monitored using an ELISA assay.

Example 4 Disease Modifying Activity of hESC-RPE in Animal Models ofRetinal Dystrophy

hESC-RPE can rescue photoreceptors and visual function. hESC-RPE cellstransplanted into dystrophic RCS rat retina survived in the subretinalspace, continued their maturation in vivo, and phagocytosed ROS. Someapproaches favored subretinal injection of hESC-RPE, our approach is togrow hESC-derived RPE on biocompatible substrates and implant these“patches” into the subretinal space in order to reestablish a functionalRPE-photoreceptor interface. Although injected cell suspensions may beeasier to perform, delivery of cells on a substrate that mimics Bruch'smembrane is used in several embodiments. There are two main reasons forthis change of approach. Firstly, the aged macula contains both agedBruch's membrane as well as aged RPE cells, and this aged membrane doesnot support attachment and growth of RPE cells. Secondly, a largeproportion of injected cells will ultimately be lost in the process dueto mechanical, or apoptotic processes; a phenomenon that has beendemonstrated in cell-suspension injection studies. In contrast, hESC-RPEgrown on polylactic glycolic acid (PLGA; a biodegradable polymer);transplanted into the subretinal space of immuno-suppressed RCS rats,can be precisely imaged by fundus photography and OCT. See FIG. 13. Inaddition, high resolution imaging is performed and used to evaluate theplacement and integrity of subretinal grafts. High speed spectral domainOCT technology (SDOCT), imaging allows for detailed three-dimensionalimaging of rodent retina and visualization of various layers. See, forexample, FIG. 16.

The substrate is configured to degrade entirely within 90 days ofimplantation. Three weeks after transplantation, eyes were processed forpathologic examination. Immunostaining using the human specific markerTRA-1-85 revealed survival of the transplanted hESC-RPE cells in thesubretinal space of dystrophic RCS rats and preservation of theoverlying photoreceptors in the outer nuclear layer (ONL) compared toadjacent retina, control eyes or polymer-alone injections. See FIG. 14.

Example 5 Treatment of Age Related Macular Degeneration

Age related macular degeneration is a condition found in elderly adultsin which the macula area of the retina suffers thinning, atrophy andbleeding. This results in the loss of vision in the central area ofvision, particularly an inability to see fine details. AMD is classifiedas either dry (non-neovascular) or wet (neovascular), with wet-AMDtypically leads to more serious vision loss. Dry macular degeneration isdiagnosed when yellowish spots known as drusen begin to accumulate fromdeposits or debris from deteriorating tissue primarily in the area ofthe macula. Gradual central vision loss may occur with dry maculardegeneration. Dry AMD can progress to wet AMD, in which new bloodvessels grow beneath the retina and leak blood and fluid. The leakageand pressure build-up damage light-sensitive retinal cells(photoreceptors), which either lose function or die, thereby leading toblind spots in central vision.

RPE transplantation strategies for AMD have been developed over the past20 years, with the goal of re-establishing the critical interactionbetween the RPE and the photoreceptor. Advances have come from a largebody of animal work and more recently a number of human trials using thepatient's own cells. However, current approaches are hampered bycomplexities in surgical procedure and limitations in cell supply andquality. The most compelling evidence from over 260 homologous andautologous RPE transplants, mostly for neovascular AMD, comes frommacular translocation studies. This complex and risky surgical procedureinvolves detaching and moving the retina so that the damaged maculararea of the retina is put into contact with healthy RPE. Although notstrictly RPE transplantation, it is functionally an autologous RPEtransplantation.

Other strategies for autologous RPE transplantation have evolved fromsubmacular RPE-choroid pedicle flap rotation to sub macularRPE-choroids-free graft transposition, submacular injection ofsuspension of RPE cells from the peripheral fundus, and currently,submacular insertion of RPE-choroid patch graft from the peripheralfundus. The latter technique involves harvesting an RPE-choroid patchgraft from the periphery followed by insertion under the macula and isbeing used currently by several European groups.

Although these results are encouraging in that vision is restored inmany cases, they point out the complexity of procedures that require twolarge incisions in the retina; the first to harvest and the second toimplant. Such large retinal incisions (retinotomies) are associated withhigh risk of retinal detachment. The approach of using cell suspensionshas the added drawback that unattached cells can easily escape into thevitreous cavity, leading to proliferation and scarring on the surface ofthe inner retina, resulting in complex and often irreparable retinaldetachments (proliferative vitreoretinopathy (PVR).

RPE cells have also been transplanted into the brain in clinical trialsfor Parkinson's disease, since they have been shown to produce dopamine.Relevant to several embodiments disclosed herein, these studies did notdetect any tumor formation by RPE. In adult humans, RPE do not replicateextensively, and reports of spontaneously occurring RPE-derived tumorsare extremely rare.

Substrates as disclosed herein are used to deliver RPE cells to thesub-retinal space of subjects having AMD to treat the vision loss byfacilitating interaction of the RPE cells with the photoreceptors,thereby supporting the photoreceptors and preventing additional loss offunction or death of the photoreceptors.

In vivo studies in rat models of AMD are performed. Substrates seededwith RPE cells are delivered to treated animals and compared to diseasedrats receiving sham (e.g. empty) substrates or sham operated normalrats.

Clinical follow-up and characterization is performed on both treatedrats and controls. Clinical assessment is done using fundus angiography(FA) (to assess vasculature health), fundus photography, fundusautofluorescence (FAF), optokinetic nystagmus (OKN), electroretinography(ERG), and multifocal ERG (mfERG). Histopathologic assessment of animalsis performed for evidence of local tumor/teratoma formation, maintenanceof a differentiated RPE phenotype of the transplanted cells with lack ofcell proliferation (immunohistochemistry using cell cycle specificantibodies), migration of cells away from the monolayer, cell loss ordamage to adjacent photoreceptors or Bruch's membrane/choroid complex,and inflammation (macrophages, T-cells), or reaction to the substrate.Transplanted hESC-RPE will be identified by monitoring expression of ahuman marker protein (TRA-1-85). The need for immunosuppression isevaluated by measuring expression of MHC Class I and II in vitro (withand without activation by interferon-gamma) and in vivo byimmunohistochemistry. Treated rats exhibit improved clinical assessmentwith respect to one or more of the parameters measured.

Example 6 Generation of Substrates Containing Stem Cells

As discussed above, in several embodiments, hESC-RPE can bedifferentiated into polarized monolayers on biodegradable andnon-biodegradable substrates In several embodiments, cells are pre-grownon a substrate that is eventually incorporated into a three dimensionalsubstrate cage for implantation. A sheet of suitable substrate, such asa polycaprolactone and polyethylene glycol (PCL-PEG) co-polymer ispolymerized at the desired thickness. While polymerizing, the co-polymeris optionally spin-coated on a coverslip to tailor the thickness. Acyclic amino acid containing PCL-PEG co-polymer is annealed to thepolymerized substrate. Aqueous media is then added to dissolve the watersoluble PEG and generate pores. The porous substrate is then sterilized.Stem cells are then cultured on the sterilized sheet to a desired celldensity. One or more pieces of cell-supporting substrate are then cutfrom the sheet, aligned and annealed with one or more additional piecesof co-polymer to generate a cell-containing three-dimensional substratecage suitable for direct implantation.

In several embodiments PLGA is used to form a biodegradable substrate.As shown in FIG. 15A, hESC-RPE grown on PLGA show well developed apicalmicrovilli after 3 weeks of culture. Other embodiments employnon-biodegradable material. hESC-RPE grown on surface-modifiednon-biodegradable parylene also show excellent attachment anddevelopment of apical microvilli (see inset of FIG. 15B) within 48 hrsof culture. While in some embodiments cells are grown on a substrateprior to fabrication of the final substrate, in other embodiments, cellsare deployed into a fully fabricated substrate prior to implantation.

Example 7 Generation of Substrates Configured to Receive Stem Cells

As discussed above, in several embodiments, substrate cages arefabricated that can later have one or more varieties of stem cellsdeployed within the substrate cages prior to implantation in a targettissue. Two molds are created that correspond to a top (apical) andbottom (basal) portion of the desired three-dimensional substrate cages.The molds are configured to yield an inner lumen upon the assembly ofthe two portions of the substrate cages and an access means to deliverystem cells to the lumen post-assembly. A polymer is polymerized withineach portion of the mold. Upon polymerization, the polymer is removedfrom its mold and exposed to an aqueous media to generate pores withinthe substrate. The pores are configured to retain cells within the finalsubstrate cage and still allow interaction (physical, chemical, orotherwise) between the cells a target tissue. The two polymeric portionsare then aligned and annealed to generate a three-dimensional poroussubstrate cage that is suitable to receive stem cells and then beimplanted into a recipient in order to generate a therapeutic effect insaid recipient through the interaction of the cells and the targettissue.

Example 8 Interdigitation of hESC-RPE and Pr Outer Segments in Rat Eye

Using a parylene substrate comprising at least one planar cell-growthsurface, interdigitation of H9 hESC-RPE with photoreceptor (PR) outersegments was demonstrated. The Royal College of Surgeons (RCS) rat is anestablished animal model for inherited retinal degeneration. The geneticdefect in RCS rats causes the inability of the retinal pigmentepithelium (RPE) to phagocytose shed photoreceptor outer segments.hESC-RPE cells were seeded onto parylene substrates and grown inaccordance with the techniques described above. Once the RPE cells hasgrown to confluency, an substrate was implanted into the RCS rat eye,with the cell growth surface juxtaposed with the outer nuclear layer ofthe photoreceptors. Control substrates (no cells) were implanted intoRCS rats for comparison.

FIG. 23A shows a stitched fundus photo of implanted parylene substratein the RCS rat eye. FIG. 23B depicts hematoxylin and eosin (H & E)staining one week post-implant in RCS rat. The lower right panel of FIG.23B shows that hESC-RPE interdigitate with PR outer segments. Themajority of the hESC-RPE are retained on the apical surface of thesubstrate. Unseeded control substrates show no such connectivity (24Blower left panel). FIG. 23C shows a cross-section of isolated orbit ofimplanted rat (1 week post-implant). FIG. 23D is a high resolutionclose-up of the parylene substrate seeded with hESC-RPE. Interdigitationand localization of RPE somas on substrate surface can be seen. See alsoFIG. 27, which is a scanning electron microscopic image of hESC-RPEseeded on a parylene substrate.

Example 9 Interdigitation of hESC-RPE and Pr Outer Segments in Rat Eye

As discussed above, interdigitation of hESC-RPE cells with the outersegments of the photoreceptors allows the functional and metabolicinteraction between the RPE cells and photoreceptors to take place. Thisinteraction supports the viability of the photoreceptors and asdiscussed in the prior examples, leads to functional vision recovery. Inthis example, the long term engraftment and interdigitation of RPE cellsin dystrophic rats was assessed by transmission electron microscopy.

As with the Examples above, substrates comprising RPE cells wereimplanted into the subretinal space of dystrophic RCS rats at postnatalday 29. The substrate use in this example was approximately 6.5 micronsthis, though as discussed above, other dimensions may be used in variousembodiments. Animals were sacrificed on post-natal day 38 (9 dayspost-surgery) and 87 (58 days after surgery). No immunosuppressivetherapy was administered to the rats.

As shown in FIG. 24, at post-implant day 9 the RPE microvilli arelocalized near the outer segment disks. At post-implant day 58, shown inFIGS. 25 and 26, interdigitation between the RPE microvilli and theouter segment disks can be seen. Thus, even at extended time-points, theRPE cells are viable and are functionally interdigitated with thephotoreceptor outer segment. This data demonstrates that RPEs implantedaccording the methods disclosed herein, and using the substratesdisclosed herein, provide long-term functional engraftment of RPEs inthe eye.

Example 10 Implanted Cell-Seeded Substrates Restore Function

As discussed herein, delivery of cells to a target site requires notonly that the cells actually reach the target site, but are viable andfunctional at the target site. Preferably, the duration of viability andfunctionality are sufficient to provide a noticeable therapeutic effect.H9 hESC-RPE were seeded onto a 0.3 micron thin film parylene substrate(with 6.5 micron (height) support features on the basal portion) asdescribed herein. The cell-seeded substrate was surgically introducedinto the sub-retinal space of a RCS rat in accordance with severalembodiments above. As shown in FIGS. 28A-28C, the parylene substrate canbe identified by the white arrow. As discussed above, the design of thesubstrate provides support for the substrate itself, and additionallyprotects the cells during the implantation procedure. Moreover, thesubstrate is designed to allow the reciprocal exchange of nutrientsbetween the RPE cells seeded on the substrate and the rich blood supplyof the choroid. FIG. 28A shows staining for TRA-1-85, which is a humanantigen marker, which shows that the cells seeded on the substrate arepresent in an intact monolayer. The staining also confirms that thecells that make up the monolayer are of human origin. FIG. 28B depictsstaining for RPE65, which is a protein involved in the process of visualpigment regeneration in the PR cells. The presence of RPE65 shows notonly that the implanted RPE cells are viable (at 2 monthspost-implantation), but also that they are functional and active in thenormal processes associated with phototransduction. FIG. 28C shows DAPI(a non-specific cellular marker) stain overlaying with the TRA-1-85 andREP65. FIGS. 29A-29C show an additional series of immunofluorescentimages from another experiment performed as described above. Asdiscussed above, these images show that the monolayer of seeded RPEcells is both intact and functional 2 months post-implantation.

Additional experiments were performed in order to determine the degreeof visual function restored following implantation of RPE-seededsubstrates. RCS rats were received either a bolus injection of RPE cellsor a cell-seeded parylene implant as disclosed herein at post-natal day28-32. FIGS. 30 and 31 depict data collected from subsequent OKNtesting. In the OKN testing, subjects (rats in these experiments) areplaced in a chamber surrounded by video monitors that generate a patternof black and white columns that move around the subject. The time thatthe subject spends tracking the visual image is indicative of the visualfunction of the subject (e.g., more tracking time is associated greatervisual function). FIG. 30 depicts OKN data collected from normal,untreated transgenic blind mice, and transgenic blind mice treated withH9 hESC-RPE injected into the sub-retinal space in a cell suspension (nosupporting substrate). As expected, normal rats had the greatesttracking time as compared to both treated and untreated blind rats(normal were tested only out to post-natal day 45). At day post-natalday 40, the treated and untreated blind rats did not significantlydiffer in their visual function. However, between post-natal day 40 andpost-natal day 45, the treated rats maintained approximately the samevisual function. In contrast, the untreated rats experienced thebeginning of a decline in visual function, which continued throughpost-natal day 60. Between post-natal day 45 and post-natal day 60, thetreated rats showed a small but insignificant decline in visualfunction, but still had discernibly improved function as compared tountreated rats.

FIG. 31 shows OKN data from normal, untreated transgenic blind mice, andtransgenic blind mice treated with H9 hESC-RPE seeded onto a parylenesubstrate and implanted in the sub-retinal space. Again, in the earlypost-implantation period, the various groups were not significantlydifferent in their tracking time. At post-natal day 45, the normal rats,as expected, had substantially greater tracking time as compared tountreated and treated rats. While the rats treated with substrates didnot show a significant difference in visual function as compared tountreated rats, past post-natal day 45, the treated animals showedrepeated, significant improvements in visual function. Not only doesthis data show a clear improvement in visual function as a result ofcell-seeded substrate implantation show as compared to untreatedanimals, but the improvement is also markedly greater than when an RPEcell suspension is administered.

For example, the head tracking time at post-natal day 60 in mice treatedwith a bolus injection was approximately 12 seconds, while in contrast,the animals treated with a cell-seeded substrate tracked images visuallyfor nearly 30 seconds. Moreover, the trend of increasing tracking timein the animals treated with a cell-seeded substrate suggests thatgreater improvement would be realized at later time points.

FIG. 32 is an additional depiction of the post-implantation RPE cellsthat have functionally interdigitated with the host photoreceptors andare metabolically active. As shown, the substrate is positioned at thebottom of the figure. Positioned on the substrate apical surface are theseeded RPE cells. Shown at the top portion of the figure are theresident photoreceptors. White arrow heads depict outer segments (beingshed from the PR) that stain positive for rhodopsin. White arrows withinthe RPE layer depict phagosomes within the RPE that contain rhodopsinpositive fragments of out segments. This demonstrates that the RPE cellsare viable, stable and functionally active in that they are taking upthe shed outer segments of the RPE, one of the normal functions ofnative RPE cells.

Thus, in several embodiments, the substrates disclosed herein not onlyprovide a surface for the formation of a monolayer of seeded RPE cells,but protect the cells during implantation into the eye of a subject, andalso support the viability of the cells post-implantation. The design ofthe substrate is such that nutrients can still reach the seeded RPEcells, but the substrate provides sufficient support to allow the cellsto maintain a monolayer in vivo. Moreover, the continued viability ofthe cells contributes to the overall restoration of visual function, asthe seeded RPE cells functionally replace the dead or damaged RPE cells,as evidenced by their uptake of shed outer segments from the PR cells.

Moreover, in several embodiments, specialized surgical methods toimplant such substrates seeded with cells are used. These surgicalprocedures not only allow placement of a substrate that is specific to aparticular subject, but also allow for the placement of one, two, ormore substrates, depending on the severity of damage to the oculartissue of the subject.

Additionally, substrates and methods as disclosed herein are useful forthe treatment of a variety of outer retinal dystrophies. Not only arethe substrates disclosed herein suitable for implantation into variousplaces of the retina, their design which enables nutrients to reach thecells seeded thereon, the substrates are suitable for supporting thegrowth and function of a wide variety of cell types. By way of exampleonly, substrates as disclosed herein could, in some embodiments, bemanufactured to be seeded with photoreceptors and implanted in order totreat retinitis pigmentosa.

Various modifications and applications of embodiments of the inventionmay be performed, without departing from the true spirit or scope of theinvention. Further, the disclosure herein of any particular feature,aspect, method, property, characteristic, quality, attribute, element,or the like in connection with an embodiment can be used in all otherembodiments set forth herein. Method steps disclosed herein need not beperformed in the order set forth. It should be understood that theinvention is not limited to the embodiments set forth herein forpurposes of exemplification, but is to be defined only by a reading ofthe appended claims, including the full range of equivalency to whicheach element thereof is entitled.

What is claimed is:
 1. A substrate for cellular therapy to treatdiseased or damaged ocular tissue, comprising: a solid planar parylenesubstrate configured for implantation into a human eye, the substratecomprising a long axis and a short axis; and a distinct parylene apicalsurface for the growth of a population of human embryonic stemcell-derived RPE cells, and a distinct parylene basal surface; whereinthe apical surface has a thickness ranging from about 0.1 to about 5microns allowing passage of molecules having a molecular weight of up toabout 75 kilodaltons and has an integrally formed continuous raised lippositioned around the perimeter to reduce migration of human stemcell-derived RPE cells off the apical surface; and wherein the basalsurface comprises a plurality of supporting protrusions, the supportingprotrusions being periodic, columnar in shape, and runninglongitudinally along the length of the long axis of the basal surface ofthe substrate and the basal surface is configured to prevent the growthof blood vessels through said basal surface to said apical surface. 2.The substrate of claim 1, wherein the parylene of said apical surface isselected from the group consisting of parylene A, parylene AM, ammoniatreated parylene, and parylene C.
 3. The substrate of claim 1, whereinthe parylene of said inhomogeneous basal surface comprises a polymerselected from the group consisting of parylene A, parylene AM, ammoniatreated parylene, and parylene C.
 4. The substrate of claim 1, whereinone or more of said apical surface and said inhomogeneous basal surfaceis treated with one or more of matrigel, poly-L-dopamine, vitronectin,or retronectin.
 5. The substrate of claim 1, wherein the height of saidsupporting protrusions ranges from about 1 μm to about 500 μm.
 6. Thesubstrate of claim 1, wherein one or more of said parylene apicalsurface and said parylene inhomogeneous basal surface areoxygen-treated.
 7. The substrate of claim 1, wherein said continuousraised lip has a height ranging from about 10 to about 1000 microns. 8.The substrate of claim 1, wherein said continuous raised lip has a widthranging from about 10 to about 1000 microns.
 9. The substrate of claim1, wherein the long axis and the short axis of parylene substrate eachrange from about 0.3 mm to about 7 mm.
 10. The substrate of claim 1,wherein said parylene inhomogeneous basal surface is treated withvitronectin.
 11. The substrate of claim 1, wherein said parylene apicalsurface is treated with vitronectin.
 12. The substrate of claim 1,wherein the damaged or diseased ocular tissue comprises ocular tissueafflicted with an outer retinal degenerative disease is selected fromthe group consisting of dry AMD, wet AMD, Stargardt's disease, Leber'sCongenital Ameurosis, and retinitis pigmentosa.
 13. The substrate ofclaim 1, wherein the substrate is suitable for implantation into thesubretinal space of the eye of a subject.
 14. The substrate claim 1,wherein upon implantation into the human eye, RPE cells on saidsubstrate functionally interdigitate with the outer segments of thephotoreceptors of the eye.
 15. A substrate for cellular therapy to treatdiseased or damaged ocular tissue, comprising: a solid planar parylenesubstrate configured for implantation into a human eye, the substratecomprising a long axis and a short axis; and a distinct parylene apicalsurface for the growth of a population of human embryonic stemcell-derived RPE cells, and a distinct parylene basal surface; whereinthe apical surface has a thickness ranging from about 0.1 to about 5microns allowing passage of molecules having a molecular weight of up toabout 75 kilodaltons; and wherein the basal surface comprises aplurality of supporting protrusions, the supporting protrusions beingperiodic, columnar in shape, and running longitudinally along the lengthof the long axis of the basal surface of the substrate, the supportingprotrusions providing structural rigidity along the long axis andpermitting flexibility along the shorter axis, and the basal surface isconfigured to prevent the growth of blood vessels through said basalsurface to said apical surface.
 16. The substrate of claim 15, whereinthe parylene of said apical surface is selected from the groupconsisting of parylene A, parylene AM, ammonia treated parylene, andparylene C.
 17. The substrate of claim 15, wherein the parylene of saidinhomogeneous basal surface comprises a polymer selected from the groupconsisting of parylene A, parylene AM, ammonia treated parylene, andparylene C.
 18. The substrate of claim 15, wherein one or more of saidapical surface and said inhomogeneous basal surface is treated with oneor more of matrigel, poly-L-dopamine, vitronectin, or retronectin. 19.The substrate of claim 15, wherein the height of said supportingprotrusions ranges from about 1 μm to about 500 μm.
 20. The substrate ofclaim 15, wherein the long axis and the short axis of parylene substrateeach range from about 0.3 mm to about 7 mm.
 21. The substrate of claim15, wherein said parylene inhomogeneous basal surface is treated withvitronectin.
 22. The substrate of claim 15, wherein said parylene apicalsurface is treated with vitronectin.
 23. The substrate of claim 1,wherein the damaged or diseased ocular tissue comprises ocular tissueafflicted with an outer retinal degenerative disease is selected fromthe group consisting of dry AMD, wet AMD, Stargardt's disease, Leber'sCongenital Ameurosis, and retinitis pigmentosa.
 24. The substrate ofclaim 15, wherein the substrate is suitable for implantation into thesubretinal space of the eye of a subject.